LOCUS Exported 7039 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Can be used to generate AAV virus that will express the ChR2(H134R)-EYFP channelrhodopsin fusion protein in neurons under intersectional control by Flp and Cre recombinases. ACCESSION . VERSION . KEYWORDS AAV phSyn1-FSF-FLEX-ChR2(H134R)-EYFP-WPRE-bGHpA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7039) AUTHORS Madisen L, Garner AR, Shimaoka D, Chuong AS, Klapoetke NC, Li L, van der Bourg A, Niino Y, Egolf L, Monetti C, Gu H, Mills M, Cheng A, Tasic B, Nguyen TN, Sunkin SM, Benucci A, Nagy A, Miyawaki A, Helmchen F, Empson RM, Knopfel T, Boyden ES, Reid RC, Carandini M, Zeng H TITLE Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. JOURNAL Neuron. 2015 Mar 4;85(5):942-58. doi: 10.1016/j.neuron.2015.02.022. PUBMED 25741722 REFERENCE 2 (bases 1 to 7039) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7039 /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 177..624 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" protein_bind 637..670 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" polyA_signal 716..764 /note="synthetic polyadenylation signal" polyA_signal 773..1249 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(972..991) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" primer_bind complement(1284..1303) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 1328..1376 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" protein_bind 1536..1569 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" protein_bind 1584..1617 /label=lox71 /bound_moiety="Cre recombinase" /note="Left element (LE) mutant of loxP (Araki et al., 2010). Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(1717..2433) /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1759..1780) /label=EGFP-C /note="EGFP, forward primer" primer_bind 2108..2127 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2367..2388 /label=EGFP-N /note="EGFP, reverse primer" regulatory 2430..2439 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS complement(2449..3375) /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 3372..3381 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(3413..3446) /label=loxJTZ17 /bound_moiety="Cre recombinase" /note="Right element (RE) mutant of loxP (Araki et al., 2010). Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 3465..4053 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3518..3538) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3936..3947) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(4071..4088) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4077..4284 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 4302..4442 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4302..4431 /label=AAV2 ITR rep_origin 4517..4972 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4604..4623) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4814..4835 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4989..5008) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5108..5130 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5168..5186) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5254..5358 /gene="bla" /label=AmpR promoter CDS 5359..6219 /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(5577..5596) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6390..6978 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6879..6898 /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 tgcggcccga acgcgtctgc agagggcctg cgtatgagtg 181 caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac gaccgacccc 241 gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg 301 gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca 361 gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg 421 acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc 481 gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac 541 catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggagga 601 gtcgtgtcgt gcctgagagc gcagacgcgt ttcgaagaag ttcctattct ctagaaagta 661 taggaacttc cctcagcacc atggctagcg gcagcctcgg agtttgaata gatagaataa 721 aatatcttta ttttcattcc atctgtgtgt tggttttttg tgtgagatct acgggtggca 781 tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac 841 cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa 901 tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag 961 ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc 1021 aatctccgcc tcctgggttc aagcgattct cctgcctcag gctcccgagt tgttgggatt 1081 ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac 1141 catattggcc aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc 1201 caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgcctcagct 1261 aattgagtag gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc 1321 gctatgacgg caataaaaag acagaataaa acgcacgggt gttgggtcgt ttgttcataa 1381 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg 1441 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc 1501 agggctcgca gccaacgtcg gggcggcagg ccgtggaagt tcctattctc tagaaagtat 1561 aggaacttcc tcgaggttaa ctttaccgtt cgtatagcat acattatacg aagttatctg 1621 cgtcgacctt gtttaaactc cggagctagc accggtggcg cgccatttaa atggccggcc 1681 tgaattcact tgccggcggc cgctttatcc ggaacccttg tacagctcgt ccatgccgag 1741 agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg 1801 gtctttgctc agggcggact ggtagctcag gtagtggttg tcgggcagca gcacggggcc 1861 gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat 1921 gttgtggcgg atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata 1981 gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt 2041 gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc 2101 ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc 2161 ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcgggcgaa 2221 gcactgcagg ccgtagccga aggtggtcac gagggtgggc cagggcacgg gcagcttgcc 2281 ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc 2341 ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac 2401 cccggtgaac agctcctcgc ccttgctcac catggtggcg gccgcgggta ctgcgccagc 2461 ctcggcctcg tcctccacca gcgtctcgac ctcaatctca gtgccaccaa tgttcaattt 2521 ggtggtcttg cgaatgtcgc cgtggatgag gatatgctcg tggatcagca cgcgcaggta 2581 gtggccgagc agaccccagc agttcttcga catcaggtca atgatggtgt ggccgacggt 2641 ggagccgtac acgctcagga cgccgaagcc ctcggggccg aggatgaaca ggatggggaa 2701 cataccccat gatacgaaga agagccaagc catgccagtc accacctggc gacaccggcc 2761 cttcggcacg gtgtggtaac cctcgatgta ggccttggca gcgtgaaaga acgtgttagc 2821 accataacac agacccaggc agaagaagat gaccttgacg tatccggtgg ccatggcgga 2881 agtggcgccc cacacaattg tgccaatatc agacacaagc agacccatgg tgcgcctgct 2941 gtagtcgttg gacaagcccg tcaggtttga caggcgaatg agaatgaccg ggcaggtgag 3001 aagccactcg gcgtaacgca accactggac gcggtggcct gtggctagat acagcatgga 3061 cgggttctta aactcgaaga agaactcgag aatcaccttg accatctcga tagcgcacac 3121 atagatctcc tcccagccgc aggttgactt ccatgtttgg taggcgtaaa acataagcag 3181 taggatggag aagccagcag caagccattg cagcacgttc gacgccgttt gggcaccgtt 3241 tgtgccacgc gactcaatcc agcccgcgca gtaacactgg tcctcaggca caagtacaga 3301 gccattgacg actactgggt tcgttacaaa tagcagctcg cgcccaacgg cactcagggc 3361 gcctccataa tccatggtgg caaggccggc caggatttaa atggcgcgcc aaataaattg 3421 ctataatgta tgctatacga agttatcact agtcgcttat cgataatcaa cctctggatt 3481 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 3541 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 3601 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 3661 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 3721 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 3781 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 3841 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 3901 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 3961 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 4021 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgcctaggg cctcgactgt 4081 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 4141 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 4201 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 4261 agacaatagc aggcatgctg gggattaatt aattgggccg caggaacccc tagtgatgga 4321 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 4381 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca 4441 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg 4501 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 4561 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 4621 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 4681 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat 4741 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 4801 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc 4861 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 4921 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc 4981 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 5041 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 5101 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 5161 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 5221 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 5281 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 5341 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 5401 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 5461 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 5521 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 5581 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 5641 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 5701 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 5761 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 5821 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 5881 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 5941 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 6001 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 6061 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 6121 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 6181 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 6241 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 6301 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 6361 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 6421 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 6481 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 6541 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 6601 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 6661 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 6721 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 6781 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 6841 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 6901 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 6961 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 7021 tggccttttg ctcacatgt //