LOCUS Exported 6626 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Intersectional viral expression of mCherry in cells expressing Flp AND NOT Cre. ACCESSION . VERSION . KEYWORDS pAAV-Ef1a-Coff/Fon-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6626) 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 6626) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6626 /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." regulatory 1550..1559 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind complement(1701..1719) /label=mCherry-R /note="mCherry, reverse primer" protein_bind complement(1918..1951) /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(2002..2035) /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(2446..2465) /label=mCherry-F /note="mCherry, forward primer" misc_feature 2753..3341 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2806..2826) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3224..3235) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3373..3849 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3572..3591) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 3889..4029 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3889..4018 /label=AAV2 ITR rep_origin 4104..4559 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4191..4210) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4401..4422 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4576..4595) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4695..4717 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4755..4773) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4841..4945 /gene="bla" /label=AmpR promoter CDS 4946..5806 /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(5164..5183) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5977..6565 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6466..6485 /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 tatactagtg ccaccatggt 1561 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca 1621 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc 1681 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc 1741 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc 1801 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat 1861 gaacttcgag gacggcggcg tggtgaccgt gacccaggtg agtacaggag ggctagcata 1921 acttcgtata aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta 1981 ttctttagat aggtgcaaag aataacttcg tataaggtat actatacgaa gttatggtac 2041 cgaagttcct attcttcaaa tagtatagga acttcaattg ttattggtgc aaagaatcac 2101 ttccttaagt ctttatactg cccaggaagt tcctattctt caaaaggtat aggaacttcg 2161 gcgcgcctta cttgtacagc tcgtccatgc cgccggtgga gtggcggccc tcggcgcgtt 2221 cgtactgttc cacgatggtg tagtcctcgt tgtgggaggt gatgtccaac ttgatgttga 2281 cgttgtaggc gccgggcagc tgcacgggct tcttggcctt gtaggtggtc ttgacctcag 2341 cgtcgtagtg gccgccgtcc ttcagcttca gcctctgctt gatctcgccc ttcagggcgc 2401 cgtcctcggg gtacatccgc tcggaggagg cctcccagcc catggtcttc ttctgcatta 2461 cggggccgtc ggaggggaag ttggtgccgc gcagcttcac cttgtagatg aactcgccgt 2521 cctgcaggga ggagtcctgt gggacgacat gacttaacca gttaaccatg ttctctgaac 2581 atgagaaggg ctggccactc tccagagctc gaagttccta tactatttga agaataggaa 2641 cttcttgcct taacccagaa attatcactg ttattcttta gaatggtgca aagagaagtt 2701 cctatacctt ttgaagaata ggaacttcga attcgatatc aagcttatcg ataatcaacc 2761 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 2821 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 2881 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 2941 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 3001 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 3061 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3121 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt 3181 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 3241 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 3301 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct 3361 cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa 3421 gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct 3481 gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa 3541 gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg 3601 gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag 3661 cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt 3721 tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg 3781 atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc 3841 cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag 3901 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 3961 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct 4021 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 4081 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 4141 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 4201 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 4261 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt 4321 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 4381 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 4441 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 4501 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt 4561 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc 4621 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca 4681 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg 4741 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 4801 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 4861 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 4921 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 4981 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 5041 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 5101 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 5161 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 5221 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 5281 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 5341 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 5401 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 5461 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 5521 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 5581 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 5641 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 5701 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 5761 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 5821 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 5881 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 5941 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 6001 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 6061 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 6121 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 6181 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 6241 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 6301 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 6361 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 6421 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 6481 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 6541 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 6601 cttttgctgg ccttttgctc acatgt //