LOCUS Exported 7166 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Intersectional viral expression of GCaMP6F in cells expressing Cre AND NOT Flp. ACCESSION . VERSION . KEYWORDS pAAV-Ef1a-Con/Foff 2.0-GCaMP6F SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7166) AUTHORS Fenno LE, Ramakrishnan C, Kim YS, Evans KE, Lo M, Vesuna S, Inoue M, Cheung KYM, Yuen E, Pichamoorthy N, Hong ASO, Deisseroth K TITLE Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals. JOURNAL Neuron. 2020 Jun 20. pii: S0896-6273(20)30432-3. doi: 10.1016/j.neuron.2020.06.003. PUBMED 32574559 REFERENCE 2 (bases 1 to 7166) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7166 /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=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 1510..1543 /label=loxN /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GTATACCT). loxN sites are compatible with each other, but incompatible with loxP or lox2272 sites." primer_bind complement(1636..1657) /label=EGFP-C /note="EGFP, forward primer" CDS complement(1870..1926) /codon_start=1 /product="calmodulin-binding peptide from avian smooth muscle myosin light chain kinase" /label=calmodulin-binding peptide /translation="RRKWNKTGHAVRAIGRLSS" regulatory 1938..1947 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(1954..1987) /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(2038..2071) /label=loxN /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GTATACCT). loxN sites are compatible with each other, but incompatible with loxP or lox2272 sites." protein_bind 2078..2111 /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)" primer_bind complement(2305..2326) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2566..2585) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" protein_bind complement(3151..3184) /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)" misc_feature 3293..3881 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3346..3366) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3764..3775) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3913..4389 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4112..4131) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4429..4569 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4429..4558 /label=AAV2 ITR rep_origin 4644..5099 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4731..4750) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4941..4962 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5116..5135) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5235..5257 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5295..5313) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5381..5485 /gene="bla" /label=AmpR promoter CDS 5486..6346 /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(5704..5723) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6517..7105 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7006..7025 /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 gatccataac ttcgtatagg 1441 atactttata cgaagttatg tagctggagc aatttgaaaa gattgttgta ttacctctag 1501 ctgcgcttaa taacttcgta tagtatacct tatacgaagt tatgtcgacc ctcctgtact 1561 caccttgctc accatgctcc ctccggtacc gcccttgtac agctcgtcca tgccgagagt 1621 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc 1681 tttcgaaagt ttggactgca cgctcaggta gtggttgtcg ggcagcagca cggggccgtc 1741 gccgatgggg gtgttctgct ggtagtggta ggcgagctgc acgccgccgt cctcgatgtt 1801 gtggcggatc ttgaagttcg ccttgatgcc gttcttctgc ttgtcggcct tgatatagac 1861 gttctcgagt gagctcagcc gacctatagc tctgactgcg tgacctgtct tattccactt 1921 acgacgtgat gagtccacca tggtggcact agtataactt cgtataaagt atcctatacg 1981 aagttatttg ccttaaccca gaaattatca ctgttattct ttagataggt gcaaagaata 2041 acttcgtata aggtatacta tacgaagtta tggtaccgaa gttcctattc tctagaaagt 2101 ataggaactt cgcagaatgg tagctggatt gtagctgcta ttagcaatat gaaacctctt 2161 agaagttcct attcttcaaa aggtatagga acttcggcgc gcctggagag tggccagccc 2221 ttctcatgtt cagagaacat ggttaactgg ttaagtcatg tcgtcccaca gggcgaggag 2281 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 2341 ttcagcgtgt ccggcgaggg tgagggcgat gccacctacg gcaagctgac cctgaagttc 2401 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 2461 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 2521 gccatgcccg aaggctacat ccaggagcgc accatcttct tcaaggacga cggcaactac 2581 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 2641 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacctgccg 2701 gaccaactga ctgaagagca gatcgcagaa tttaaagagg aattctccct atttgacaag 2761 gacggggatg ggacaataac aaccaaggag ctggggacgg tgatgcggtc tctggggcag 2821 aaccccacag aagcagagct gcaggacatg atcaatgaag tagatgccga cggtgacggc 2881 acaatcgact tccctgagtt cctgacaatg atggcaagaa aaatgaaata cagggacacg 2941 gaagaagaaa ttagagaagc gttcggtgtg tttgataagg atggcaatgg ctacatcagt 3001 gcagcagagc ttcgccacgt gatgacaaac cttggagaga agttaacaga tgaagaggtt 3061 gatgaaatga tcagggaagc agacatcgat ggggatggtc aggtaaacta cgaagagttt 3121 gtacaaatga tgacagcgaa gtgagctagc gaagttccta tactttctag agaataggaa 3181 cttctaagca gacaatcttg taatacatca gcagctagta gattgctcca gctagaagtt 3241 cctatacctt ttgaagaata ggaacttcga attcgatatc aagcttatcg ataatcaacc 3301 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 3361 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 3421 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 3481 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 3541 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 3601 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3661 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt 3721 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 3781 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 3841 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct 3901 cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa 3961 gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct 4021 gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa 4081 gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg 4141 gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag 4201 cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt 4261 tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg 4321 atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc 4381 cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag 4441 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 4501 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct 4561 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 4621 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 4681 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 4741 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 4801 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt 4861 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 4921 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 4981 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 5041 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt 5101 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc 5161 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca 5221 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg 5281 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 5341 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 5401 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 5461 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 5521 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 5581 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 5641 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 5701 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 5761 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 5821 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 5881 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 5941 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 6001 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 6061 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 6121 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 6181 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 6241 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 6301 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 6361 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 6421 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 6481 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 6541 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 6601 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 6661 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 6721 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 6781 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 6841 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 6901 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 6961 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 7021 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 7081 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 7141 cttttgctgg ccttttgctc acatgt //