LOCUS Exported 7154 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Intersectional viral expression of GCaMP6F in cells expressing both Cre AND Flp. ACCESSION . VERSION . KEYWORDS pAAV-Ef1a-Con/Fon-GCaMP6F SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7154) 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 7154) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7154 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 231..1409 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 462..1400 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1357..1377 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 1416..1449 /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 1500..1533 /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(1626..1647) /label=EGFP-C /note="EGFP, forward primer" CDS complement(1860..1916) /codon_start=1 /product="calmodulin-binding peptide from avian smooth muscle myosin light chain kinase" /label=calmodulin-binding peptide /translation="RRKWNKTGHAVRAIGRLSS" regulatory 1928..1937 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(1942..1975) /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(2026..2059) /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 2751..2770 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3010..3031 /label=EGFP-N /note="EGFP, reverse primer" misc_feature 3281..3869 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3334..3354) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3752..3763) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3901..4377 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4100..4119) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4417..4557 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4417..4546 /label=AAV2 ITR rep_origin 4632..5087 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4719..4738) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4929..4950 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5104..5123) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5223..5245 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5283..5301) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5369..5473 /gene="bla" /label=AmpR promoter CDS 5474..6334 /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(5692..5711) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6505..7093 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6994..7013 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga 181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg 241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg 301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt 361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg 421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt 481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct 541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga 601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct 661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt 721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc 781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg 841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac 901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc 961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag 1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct 1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg 1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga 1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc 1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg 1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa 1381 gtttttttct tccatttcag gtgtcgtgag gatccataac ttcgtatagg atactttata 1441 cgaagttatg tagctggagc aatttgaaaa gattgttgta ttacctctag ctgcgcttaa 1501 taacttcgta tagtatacct tatacgaagt tatgtcgacc ctcctgtact caccttgctc 1561 accatgctcc ctccggtacc gcccttgtac agctcgtcca tgccgagagt gatcccggcg 1621 gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc tttcgaaagt 1681 ttggactgca cgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg 1741 gtgttctgct ggtagtggta ggcgagctgc acgccgccgt cctcgatgtt gtggcggatc 1801 ttgaagttcg ccttgatgcc gttcttctgc ttgtcggcct tgatatagac gttctcgagt 1861 gagctcagcc gacctatagc tctgactgcg tgacctgtct tattccactt acgacgtgat 1921 gagtccacca tggtggctag cataacttcg tataaagtat cctatacgaa gttatttgcc 1981 ttaacccaga aattatcact gttattcttt agataggtgc aaagaataac ttcgtataag 2041 gtatactata cgaagttatg gtaccgaagt tcctattctt caaatagtat aggaacttca 2101 attgttattg gtgcaaagaa tcacttcctt aagtctttat actgcccagg aagttcctat 2161 tcttcaaaag gtataggaac ttcggcgcgc ctcacttcgc tgtcatcatt tgtacaaact 2221 cttcgtagtt tacctgacca tccccatcga tgtctgcttc cctgatcatt tcatcaacct 2281 cttcatctgt taacttctct ccaaggtttg tcatcacgtg gcgaagctct gctgcactga 2341 tgtagccatt gccatcctta tcaaacacac cgaacgcttc tctaatttct tcttccgtgt 2401 ccctgtattt catttttctt gccatcattg tcaggaactc agggaagtcg attgtgccgt 2461 caccgtcggc atctacttca ttgatcatgt cctgcagctc tgcttctgtg gggttctgcc 2521 ccagagaccg catcaccgtc cccagctcct tggttgttat tgtcccatcc ccgtccttgt 2581 caaataggga gaattcctct ttaaattctg cgatctgctc ttcagtcagt tggtccggca 2641 ggttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca 2701 gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt 2761 tgccgtcgtc cttgaagaag atggtgcgct cctggatgta gccttcgggc atggcggact 2821 tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg 2881 tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact 2941 tcagggtcag cttgccgtag gtggcatcgc cctcaccctc gccggacacg ctgaacttgt 3001 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct 3061 cgccctgtgg gacgacatga cttaaccagt taaccatgtt ctctgaacat gagaagggct 3121 ggccactctc catctagaga agttcctata ctatttgaag aataggaact tcttgcctta 3181 acccagaaat tatcactgtt attctttaga atagtgcaaa gagaagttcc tatacctttt 3241 gaagaatagg aacttcgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa 3301 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 3361 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc 3421 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg 3481 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 3541 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 3601 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 3661 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat 3721 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 3781 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag 3841 tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac 3901 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 3961 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 4021 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 4081 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg 4141 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg 4201 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg 4261 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct 4321 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg 4381 attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg 4441 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 4501 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc 4561 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa 4621 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 4681 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc 4741 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 4801 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc 4861 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 4921 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc 4981 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta 5041 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct 5101 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 5161 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 5221 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 5281 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 5341 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 5401 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 5461 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 5521 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 5581 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 5641 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 5701 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 5761 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 5821 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 5881 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 5941 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 6001 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 6061 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 6121 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 6181 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 6241 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 6301 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 6361 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 6421 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 6481 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 6541 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 6601 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 6661 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 6721 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 6781 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 6841 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 6901 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 6961 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 7021 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 7081 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 7141 ttttgctcac atgt //