LOCUS Exported 7251 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Cre-activated AAV expression of humanized ChR2 with H134R mutation fused to mCherry for optogenetic activation. ACCESSION . VERSION . KEYWORDS pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7251) TITLE Cre-activated AAV expression plasmids REFERENCE 2 (bases 1 to 7251) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7251 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(62..650) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(142..161) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(821..1681) /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 1444..1463 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1682..1786) /gene="bla" /label=AmpR promoter primer_bind 1854..1872 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(1910..1932) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 2032..2051 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(2068..2523) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2205..2226) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2417..2436 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 2598..2738 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 2609..2738 /label=AAV2 ITR polyA_signal 2778..3254 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind 3036..3055 /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" misc_feature 3286..3874 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" CDS 3392..3403 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind 3801..3821 /label=WPRE-R /note="WPRE, reverse primer" protein_bind 3899..3932 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 3983..4016 /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)." regulatory 4021..4030 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4027..4953 /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 PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI RKTTKLNIGGTEIEVETLVEDEAEAGAV" CDS 4966..5673 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(5108..5126) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 5376..5395 /label=mCherry-F /note="mCherry, forward primer" protein_bind complement(5685..5718) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(5769..5802) /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)." promoter complement(5833..7010) /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 5842..6779 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind complement(5865..5885) /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" repeat_region 7111..7251 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 7111..7240 /label=AAV2 ITR ORIGIN 1 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 481 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 661 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 721 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 781 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 841 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 901 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 961 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1021 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1081 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1141 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1201 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1261 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1321 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1381 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1441 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1501 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1561 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1621 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1681 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1741 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1801 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 1861 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 1921 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 1981 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 2041 agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg 2101 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc 2161 cttataaatc aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga 2221 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg 2281 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag 2341 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 2401 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg 2461 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg 2521 cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa 2581 ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg 2641 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg 2701 cagagaggga gtggccaact ccatcactag gggttcctgc ggccgctcgg tccgcacgtg 2761 gttacctaca aaatcagaag gacagggaag ggagcagtgg ttcacgcctg taatcccagc 2821 aatttgggag gccaaggtgg gtagatcacc tgagattagg agttggagac cagcctggcc 2881 aatatggtga aaccccgtct ctaccaaaaa aacaaaaatt agctgagcct ggtcatgcat 2941 gcctggaatc ccaacaactc gggaggctga ggcaggagaa tcgcttgaac ccaggaggcg 3001 gagattgcag tgagccaaga ttgtgccact gcactccagc ttggttccca atagaccccg 3061 caggccctac aggttgtctt cccaacttgc cccttgctcc ataccacccc cctccacccc 3121 ataatattat agaaggacac ctagtcagac aaaatgatgc aacttaattt tattaggaca 3181 aggctggtgg gcactggagt ggcaacttcc agggccagga gaggcactgg ggaggggtca 3241 cagggatgcc acccgtagat ctctcgagca gcgctcggta tcgatgcggg gaggcggccc 3301 aaagggagat ccgactcgtc tgagggcgaa ggcgaagacg cggaagaggc cgcagagccg 3361 gcagcaggcc gcgggaagga aggtccgctg gattgagggc cgaagggacg tagcagaagg 3421 acgtcccgcg cagaatccag gtggcaacat aggcgagcag ccaaggaaag gacgatgatt 3481 tccccgacaa caccacggaa ttgtcagtgc ccaacagccg agcccctgtc cagcagcggg 3541 caaggcaggc ggcgatgagt tccgccgtgg caatagggag ggggaaagcg aaagtcccgg 3601 aaaggagctg acaggtggtg gcaatgcccc aaccagtggg ggttgcgtca gcaaacacag 3661 tgcacaccac gccacgttgc ctgacaacgg gccacaactc ctcataaaga gacagcaacc 3721 aggatttata caaggaggag aaaatgaaag ccatacggga agcaatagca tgatacaaag 3781 gcattaaagc agcgtatcca catagcgtaa aaggagcaac atagttaaga ataccagtca 3841 atctttcaca aattttgtaa tccagaggtt gattatcgat aagcttgata tcgaattcat 3901 aacttcgtat agcatacatt atacgaagtt attctttgca ccattctaaa gaataacagt 3961 gataatttct gggttaaggc aaataacttc gtataggata ctttatacga agttatgcta 4021 gccaccatgg actatggcgg cgctttgtct gccgtcggac gcgaactttt gttcgttact 4081 aatcctgtgg tggtgaacgg gtccgtcctg gtccctgagg atcaatgtta ctgtgccgga 4141 tggattgaat ctcgcggcac gaacggcgct cagaccgcgt caaatgtcct gcagtggctt 4201 gcagcaggat tcagcatttt gctgctgatg ttctatgcct accaaacctg gaaatctaca 4261 tgcggctggg aggagatcta tgtgtgcgcc attgaaatgg ttaaggtgat tctcgagttc 4321 ttttttgagt ttaagaatcc ctctatgctc taccttgcca caggacaccg ggtgcagtgg 4381 ctgcgctatg cagagtggct gctcacttgt cctgtcatcc ttatccgcct gagcaacctc 4441 accggcctga gcaacgacta cagcaggaga accatgggac tccttgtctc agacatcggg 4501 actatcgtgt ggggggctac cagcgccatg gcaaccggct atgttaaagt catcttcttt 4561 tgtcttggat tgtgctatgg cgcgaacaca ttttttcacg ccgccaaagc atatatcgag 4621 ggttatcata ctgtgccaaa gggtcggtgc cgccaggtcg tgaccggcat ggcatggctg 4681 tttttcgtga gctggggtat gttcccaatt ctcttcattt tggggcccga aggttttggc 4741 gtcctgagcg tctatggctc caccgtaggt cacacgatta ttgatctgat gagtaaaaat 4801 tgttgggggt tgttgggaca ctacctgcgc gtcctgatcc acgagcacat attgattcac 4861 ggagatatcc gcaaaaccac caaactgaac atcggcggaa cggagatcga ggtcgagact 4921 ctcgtcgaag acgaagccga ggccggagcc gtgccagcgg ccgccgtgag caagggcgag 4981 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc 5041 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc 5101 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg 5161 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac 5221 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac 5281 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag 5341 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg 5401 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc 5461 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac 5521 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc 5581 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac 5641 tccaccggcg gcatggacga gctgtacaag taatagggcg cgccataact tcgtataatg 5701 tatgctatac gaagttatta agaggtttca tattgctaat agcagctaca atccagctac 5761 cattctgcat aacttcgtat aaagtatcct atacgaagtt attccggagt cgactctaga 5821 ggatccggta cctcacgaca cctgaaatgg aagaaaaaaa ctttgaacca ctgtctgagg 5881 cttgagaatg aaccaagatc caaactcaaa aagggcaaat tccaaggaga attacatcaa 5941 gtgccaagct ggcctaactt cagtctccac ccactcagtg tggggaaact ccatcgcata 6001 aaacccctcc ccccaaccta aagacgacgt actccaaaag ctcgagaact aatcgaggtg 6061 cctggacggc gcccggtact ccgtggagtc acatgaagcg acggctgagg acggaaaggc 6121 ccttttcctt tgtgtgggtg actcacccgc ccgctctccc gagcgccgcg tcctccattt 6181 tgagctccct gcagcagggc cgggaagcgg ccatctttcc gctcacgcaa ctggtgccga 6241 ccgggccagc cttgccgccc agggcggggc gatacacggc ggcgcgaggc caggcaccag 6301 agcaggccgg ccagcttgag actacccccg tccgattctc ggtggccgcg ctcgcaggcc 6361 ccgcctcgcc gaacatgtgc gctgggacgc acgggccccg tcgccgcccg cggccccaaa 6421 aaccgaaata ccagtgtgca gatcttggcc cgcatttaca agactatctt gccagaaaaa 6481 aagcgtcgca gcaggtcatc aaaaatttta aatggctaga gacttatcga aagcagcgag 6541 acaggcgcga aggtgccacc agattcgcac gcggcggccc cagcgcccag gccaggcctc 6601 aactcaagca cgaggcgaag gggctcctta agcgcaaggc ctcgaactct cccacccact 6661 tccaacccga agctcgggat caagaatcac gtactgcagc cagtggaagt aattcaaggc 6721 acgcaagggc cataacccgt aaagaggcca ggcccgcggg aaccacacac ggcacttacc 6781 tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca cggcgactac tgcacttata 6841 tacggttctc ccccaccctc gggaaaaagg cggagccagt acacgacatc actttcccag 6901 tttaccccgc gccaccttct ctaggcaccg gttcaattgc cgacccctcc ccccaacttc 6961 tcggggactg tgggcgatgt gcgctctgcc cactgacggg caccggagcc aattcccact 7021 cctttcaaga cctagaaggt ccattagctg caaagattcc tctctgttta aaactttatc 7081 catctttgca aagcttacgc gtgcggccgc aggaacccct agtgatggag ttggccactc 7141 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 7201 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag g //