LOCUS Exported 7256 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV expression of EF1a-driven, Cre-dependent, humanized channelrhodopsin E123T/T159C mutant fused to EYFP for optogenetic activation.. ACCESSION . VERSION . KEYWORDS pAAV-Ef1a-DIO hChR2(E123T/T159C)-EYFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7256) AUTHORS Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R, Gunaydin LA, Hyun M, Fenno LE, Gradinaru V, Yizhar O, Deisseroth K TITLE Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. JOURNAL Nat Methods. 2011 Dec 18;9(2):159-72. doi: 10.1038/nmeth.1808. PUBMED 22179551 REFERENCE 2 (bases 1 to 7256) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7256 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 225..1403 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 456..1394 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1351..1371 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 1434..1467 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind 1518..1551 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(1563..2282) /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1608..1629) /label=EGFP-C /note="EGFP, forward primer" primer_bind 1957..1976 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2216..2237 /label=EGFP-N /note="EGFP, reverse primer" regulatory 2279..2288 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS complement(2298..3224) /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 PSMLYLATGHRVQWLRYATWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGCIVWG ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI RKTTKLNIGGTEIEVETLVEDEAEAGAV" regulatory 3221..3230 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(3235..3268) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind complement(3319..3352) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 3377..3965 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3430..3450) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3848..3859) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3997..4473 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4196..4215) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4513..4653 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4513..4642 /label=AAV2 ITR rep_origin 4728..5183 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4815..4834) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5025..5046 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5200..5219) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5319..5341 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5379..5397) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5465..5569 /gene="bla" /label=AmpR promoter CDS 5570..6430 /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(5788..5807) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6601..7189 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7090..7109 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 7251..124 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" ORIGIN 1 ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc 61 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt 121 tcctgcggcc gcacgcgtaa gctttgcaaa gatggataaa gttttaaaca gagaggaatc 181 tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc 241 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt 301 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc 361 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg 421 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg 481 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc 541 agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt 601 gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc 661 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag 721 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa 781 atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg 841 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa 901 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt 961 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa 1021 gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag 1081 agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt 1141 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt 1201 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg 1261 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg 1321 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt 1381 ttcttccatt tcaggtgtcg tgaggtaccg gatcctctag agtcgactcc ggaataactt 1441 cgtataggat actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag 1501 caatatgaaa cctcttaata acttcgtata gcatacatta tacgaagtta tggcgcgccc 1561 tattacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc 1621 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg gtagctcagg 1681 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg 1741 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg 1801 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc 1861 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc 1921 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg 1981 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc 2041 tgcttcatgt ggtcggggta gcgggcgaag cactgcaggc cgtagccgaa ggtggtcacg 2101 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg 2161 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg 2221 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc 2281 atggtggcgg ccgctggcac ggctccggcc tcggcttcgt cttcgacgag agtctcgacc 2341 tcgatctccg ttccgccgat gttcagtttg gtggttttgc ggatatctcc gtgaatcaat 2401 atgtgctcgt ggatcaggac gcgcaggtag tgtcccaaca acccccaaca atttttactc 2461 atcagatcaa taatcgtgtg acctacggtg gagccataga cgctcaggac gccaaaacct 2521 tcgggcccca aaatgaagag aattgggaac ataccccagc tcacgaaaaa cagccatgcc 2581 atgccggtca cgacctggcg gcaccgaccc tttggcacag tatgataacc ctcgatatat 2641 gctttggcgg cgtgaaaaaa tgtgttcgcg ccatagcaca atccaagaca aaagaagatg 2701 actttaacat agccggttgc catggcgctg gtagcccccc acacgataca cccgatgtct 2761 gagacaagga gtcccatggt tctcctgctg tagtcgttgc tcaggccggt gaggttgctc 2821 aggtggataa ggatgacagg acaagtgagc agccaggttg catagcgcag ccactgcacc 2881 cggtgtcctg tggcaaggta gagcatagag ggattcttaa actcaaaaaa gaactcgaga 2941 atcaccttaa ccatttcaat ggcgcacaca tagatctcct cccagccgca tgtagatttc 3001 caggtttggt aggcatagaa catcagcagc aaaatgctga atcctgctgc aagccactgc 3061 aggacatttg acgcggtctg agcgccgttc gtgccgcgag attcaatcca tccggcacag 3121 taacattgat cctcagggac caggacggac ccgttcacca ccacaggatt agtaacgaac 3181 aaaagttcgc gtccgacggc agacaaagcg ccgccatagt ccatggtggc tagcataact 3241 tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc 3301 tttagaatgg tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga 3361 tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat 3421 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca 3481 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc 3541 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc 3601 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt 3661 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg 3721 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc 3781 ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta 3841 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg 3901 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc 3961 cccgcatcga taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc 4021 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata 4081 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg 4141 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat 4201 tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg 4261 gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc 4321 aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg 4381 gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta 4441 caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga 4501 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 4561 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 4621 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta 4681 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag 4741 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 4801 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 4861 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 4921 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata 4981 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 5041 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc 5101 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 5161 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc 5221 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 5281 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 5341 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 5401 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 5461 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 5521 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 5581 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 5641 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 5701 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 5761 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 5821 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 5881 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 5941 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 6001 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 6061 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 6121 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 6181 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 6241 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 6301 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 6361 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 6421 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 6481 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 6541 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 6601 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 6661 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 6721 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 6781 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 6841 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 6901 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 6961 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 7021 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 7081 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 7141 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 7201 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgca //