LOCUS Exported 6635 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Intersectional viral expression of EYFP in cells expressing Cre AND NOT Flp. ACCESSION . VERSION . KEYWORDS pAAV-Ef1a-Con/Foff 2.0-EYFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6635) 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 6635) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6635 /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 1656..1677 /label=EGFP-N /note="EGFP, reverse primer" regulatory 1719..1728 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(1735..1768) /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(1819..1852) /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 1859..1892 /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(2200..2219) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" CDS 2359..2610 /codon_start=1 /product="C-terminal fragment of mVenus for use in bimolecular fluorescence complementation (BiFC) (Kodama and Hu, 2010)" /label=VC155 /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK" primer_bind 2547..2568 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(2620..2653) /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 2762..3350 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2815..2835) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3233..3244) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3382..3858 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3581..3600) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 3898..4038 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3898..4027 /label=AAV2 ITR rep_origin 4113..4568 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4200..4219) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4410..4431 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4585..4604) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4704..4726 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4764..4782) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4850..4954 /gene="bla" /label=AmpR promoter CDS 4955..5815 /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(5173..5192) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5986..6574 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6475..6494 /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 caccttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc 1621 gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat 1681 gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggcac tagtataact 1741 tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc 1801 tttagatagg tgcaaagaat aacttcgtat aaggtatact atacgaagtt atggtaccga 1861 agttcctatt ctctagaaag tataggaact tcgcagaatg gtagctggat tgtagctgct 1921 attagcaata tgaaacctct tagaagttcc tattcttcaa aaggtatagg aacttcggcg 1981 cgcctggaga gtggccagcc cttctcatgt tcagagaaca tggttaactg gttaagtcat 2041 gtcgtcccac agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg 2101 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 2161 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 2221 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 2281 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 2341 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc 2401 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 2461 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc 2521 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 2581 atcactctcg gcatggacga gctgtacaag taagctagcg aagttcctat actttctaga 2641 gaataggaac ttctaagcag acaatcttgt aatacatcag cagctagtag attgctccag 2701 ctagaagttc ctataccttt tgaagaatag gaacttcgaa ttcgatatca agcttatcga 2761 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 2821 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 2881 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 2941 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 3001 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 3061 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 3121 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 3181 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 3241 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 3301 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg 3361 agcgctgctc gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg 3421 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc 3481 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc 3541 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg 3601 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc 3661 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt 3721 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa 3781 tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact 3841 gctcccttcc ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg 3901 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 3961 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag 4021 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 4081 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc 4141 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 4201 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 4261 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 4321 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 4381 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 4441 caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg 4501 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt 4561 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc 4621 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 4681 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 4741 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 4801 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 4861 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 4921 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 4981 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 5041 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 5101 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 5161 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 5221 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 5281 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 5341 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 5401 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 5461 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 5521 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 5581 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 5641 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 5701 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 5761 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 5821 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 5881 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 5941 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 6001 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 6061 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 6121 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 6181 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 6241 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 6301 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 6361 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 6421 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 6481 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 6541 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 6601 gttcctggcc ttttgctggc cttttgctca catgt //