LOCUS Exported 7241 bp ds-DNA circular SYN 14-FEB-2022 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 7241) TITLE Cre-activated AAV expression plasmids REFERENCE 2 (bases 1 to 7241) AUTHORS . TITLE Direct Submission JOURNAL Exported Feb 14, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7241 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 87..542 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(174..193) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 384..405 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(559..578) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 678..700 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(738..756) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 824..928 /gene="bla" /label=AmpR promoter CDS 929..1789 /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(1147..1166) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1960..2548 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2449..2468 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 2610..2739 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 2840..4018 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 3071..4009 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3966..3986 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 4049..4082 /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 4133..4166 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(4178..4885) /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(4456..4475) /label=mCherry-F /note="mCherry, forward primer" primer_bind 4725..4743 /label=mCherry-R /note="mCherry, reverse primer" CDS complement(4898..5824) /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" regulatory 5821..5830 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(5835..5868) /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(5919..5952) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 5977..6565 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6030..6050) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6448..6459) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 6597..7073 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(6796..6815) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 7113..12 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 7113..1 /label=AAV2 ITR ORIGIN 1 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 61 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 121 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt 181 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 241 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 301 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 361 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc 421 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa 481 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 541 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 601 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 661 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 721 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 781 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 841 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 901 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 961 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 1021 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 1081 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 1141 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 1201 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 1261 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 1321 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 1381 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 1441 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 1501 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 1561 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 1621 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 1681 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1741 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1801 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1861 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1921 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1981 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 2041 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 2101 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 2161 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 2221 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 2281 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2341 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2401 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 2461 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2521 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2581 ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac 2641 tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 2701 gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg cgtaagcttt 2761 gcaaagatgg ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt 2821 cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac 2881 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg 2941 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg 3001 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc 3061 cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg 3121 cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct 3181 tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 3241 tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 3301 tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 3361 tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 3421 tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 3481 ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 3541 ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 3601 gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 3661 agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 3721 aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 3781 gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 3841 ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 3901 agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 3961 cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgagg 4021 taccggatcc tctagagtcg actccggaat aacttcgtat aggatacttt atacgaagtt 4081 atgcagaatg gtagctggat tgtagctgct attagcaata tgaaacctct taataacttc 4141 gtatagcata cattatacga agttatggcg cgccctatta cttgtacagc tcgtccatgc 4201 cgccggtgga gtggcggccc tcggcgcgtt cgtactgttc cacgatggtg tagtcctcgt 4261 tgtgggaggt gatgtccaac ttgatgttga cgttgtaggc gccgggcagc tgcacgggct 4321 tcttggcctt gtaggtggtc ttgacctcag cgtcgtagtg gccgccgtcc ttcagcttca 4381 gcctctgctt gatctcgccc ttcagggcgc cgtcctcggg gtacatccgc tcggaggagg 4441 cctcccagcc catggtcttc ttctgcatta cggggccgtc ggaggggaag ttggtgccgc 4501 gcagcttcac cttgtagatg aactcgccgt cctgcaggga ggagtcctgg gtcacggtca 4561 ccacgccgcc gtcctcgaag ttcatcacgc gctcccactt gaagccctcg gggaaggaca 4621 gcttcaagta gtcggggatg tcggcggggt gcttcacgta ggccttggag ccgtacatga 4681 actgagggga caggatgtcc caggcgaagg gcagggggcc acccttggtc accttcagct 4741 tggcggtctg ggtgccctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt 4801 ggccgttcac ggagccctcc atgtgcacct tgaagcgcat gaactccttg atgatggcca 4861 tgttatcctc ctcgcccttg ctcacggcgg ccgctggcac ggctccggcc tcggcttcgt 4921 cttcgacgag agtctcgacc tcgatctccg ttccgccgat gttcagtttg gtggttttgc 4981 ggatatctcc gtgaatcaat atgtgctcgt ggatcaggac gcgcaggtag tgtcccaaca 5041 acccccaaca atttttactc atcagatcaa taatcgtgtg acctacggtg gagccataga 5101 cgctcaggac gccaaaacct tcgggcccca aaatgaagag aattgggaac ataccccagc 5161 tcacgaaaaa cagccatgcc atgccggtca cgacctggcg gcaccgaccc tttggcacag 5221 tatgataacc ctcgatatat gctttggcgg cgtgaaaaaa tgtgttcgcg ccatagcaca 5281 atccaagaca aaagaagatg actttaacat agccggttgc catggcgctg gtagcccccc 5341 acacgatagt cccgatgtct gagacaagga gtcccatggt tctcctgctg tagtcgttgc 5401 tcaggccggt gaggttgctc aggcggataa ggatgacagg acaagtgagc agccactctg 5461 catagcgcag ccactgcacc cggtgtcctg tggcaaggta gagcatagag ggattcttaa 5521 actcaaaaaa gaactcgaga atcaccttaa ccatttcaat ggcgcacaca tagatctcct 5581 cccagccgca tgtagatttc caggtttggt aggcatagaa catcagcagc aaaatgctga 5641 atcctgctgc aagccactgc aggacatttg acgcggtctg agcgccgttc gtgccgcgag 5701 attcaatcca tccggcacag taacattgat cctcagggac caggacggac ccgttcacca 5761 ccacaggatt agtaacgaac aaaagttcgc gtccgacggc agacaaagcg ccgccatagt 5821 ccatggtggc tagcataact tcgtataaag tatcctatac gaagttattt gccttaaccc 5881 agaaattatc actgttattc tttagaatgg tgcaaagaat aacttcgtat aatgtatgct 5941 atacgaagtt atgaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt 6001 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 6061 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 6121 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 6181 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 6241 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 6301 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 6361 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg 6421 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 6481 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 6541 atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt 6601 ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc 6661 ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct 6721 ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct 6781 gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca 6841 ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg 6901 gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt 6961 tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc 7021 ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt 7081 tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac 7141 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 7201 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc a //