LOCUS Exported 7071 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION An AAV packaging vector that expresses channel rhodopsin 2 (H134R) (fused to EYFP) under the EF1a promoter, which can be deactivated (deleted) by recombination between the flanking loxP sites.. ACCESSION . VERSION . KEYWORDS pOTTC292 - pAAV EF1a floxed hChR2(H134R)-EYFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7071) AUTHORS McDevitt RA, Tiran-Cappello A, Shen H, Balderas I, Britt JP, Marino RAM, Chung SL, Richie CT, Harvey BK, Bonci A TITLE Serotonergic versus nonserotonergic dorsal raphe projection neurons: differential participation in reward circuitry. JOURNAL Cell Rep. 2014 Sep 25;8(6):1857-1869. doi: 10.1016/j.celrep.2014.08.037. Epub 2014 Sep 18. PUBMED 25242321 REFERENCE 2 (bases 1 to 7071) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7071 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..141 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 12..141 /label=AAV2 ITR promoter 242..1419 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 473..1410 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1367..1387 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 1426..1459 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." regulatory 1464..1473 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1470..2396 /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 2406..2415 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2412..3131 /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2457..2478) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2718..2737) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3065..3086 /label=EGFP-C /note="EGFP, forward primer" protein_bind 3140..3173 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 3198..3786 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3251..3271) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3669..3680) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3818..4294 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4017..4036) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4334..4474 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4334..4463 /label=AAV2 ITR rep_origin 4549..5004 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4636..4655) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4846..4867 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5021..5040) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5140..5162 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5200..5218) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5286..5390 /gene="bla" /label=AmpR promoter CDS 5391..6251 /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(5609..5628) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6422..7010 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6911..6930 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt 181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat 241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg 301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag 361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc 421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc 481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat 541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccataac ttcgtatagc 1441 atacattata cgaagttatg ctagccacca tggactatgg cggcgctttg tctgccgtcg 1501 gacgcgaact tttgttcgtt actaatcctg tggtggtgaa cgggtccgtc ctggtccctg 1561 aggatcaatg ttactgtgcc ggatggattg aatctcgcgg cacgaacggc gctcagaccg 1621 cgtcaaatgt cctgcagtgg cttgcagcag gattcagcat tttgctgctg atgttctatg 1681 cctaccaaac ctggaaatct acatgcggct gggaggagat ctatgtgtgc gccattgaaa 1741 tggttaaggt gattctcgag ttcttttttg agtttaagaa tccctctatg ctctaccttg 1801 ccacaggaca ccgggtgcag tggctgcgct atgcagagtg gctgctcact tgtcctgtca 1861 tccttatccg cctgagcaac ctcaccggcc tgagcaacga ctacagcagg agaaccatgg 1921 gactccttgt ctcagacatc gggactatcg tgtggggggc taccagcgcc atggcaaccg 1981 gctatgttaa agtcatcttc ttttgtcttg gattgtgcta tggcgcgaac acattttttc 2041 acgccgccaa agcatatatc gagggttatc atactgtgcc aaagggtcgg tgccgccagg 2101 tcgtgaccgg catggcatgg ctgtttttcg tgagctgggg tatgttccca attctcttca 2161 ttttggggcc cgaaggtttt ggcgtcctga gcgtctatgg ctccaccgta ggtcacacga 2221 ttattgatct gatgagtaaa aattgttggg ggttgttggg acactacctg cgcgtcctga 2281 tccacgagca catattgatt cacggagata tccgcaaaac caccaaactg aacatcggcg 2341 gaacggagat cgaggtcgag actctcgtcg aagacgaagc cgaggccgga gccgtgccag 2401 cggccgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg 2461 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg 2521 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc 2581 cctggcccac cctcgtgacc accttcggct acggcctgca gtgcttcgcc cgctaccccg 2641 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc 2701 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg 2761 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 2821 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca 2881 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg 2941 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc 3001 ccgacaacca ctacctgagc taccagtccg ccctgagcaa agaccccaac gagaagcgcg 3061 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc 3121 tgtacaagta aggcgcgcca taacttcgta tagcatacat tatacgaagt tatgaattcg 3181 atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta 3241 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc 3301 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt 3361 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg 3421 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt 3481 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct 3541 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt 3601 cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct 3661 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc 3721 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct 3781 ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc 3841 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat 3901 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag 3961 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta 4021 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg 4081 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac 4141 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct 4201 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt 4261 acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg 4321 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 4381 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 4441 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt 4501 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta 4561 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 4621 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 4681 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 4741 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat 4801 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 4861 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc 4921 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 4981 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg 5041 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 5101 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 5161 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt 5221 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa 5281 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 5341 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 5401 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 5461 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 5521 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 5581 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 5641 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 5701 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 5761 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 5821 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 5881 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 5941 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 6001 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 6061 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 6121 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 6181 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 6241 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 6301 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 6361 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 6421 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 6481 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 6541 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 6601 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 6661 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 6721 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 6781 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 6841 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 6901 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 6961 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 7021 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t //