LOCUS Exported 7159 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Intersectional viral expression of sRGECO in cells expressing Flp AND NOT Cre. ACCESSION . VERSION . KEYWORDS pAAV-Ef1a-Coff/Fon-sRGECO SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7159) 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 7159) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7159 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 172..627 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(259..278) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 469..490 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(644..663) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 763..785 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(823..841) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 909..1013 /gene="bla" /label=AmpR promoter CDS 1014..1874 /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(1232..1251) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2045..2633 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2534..2553 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 2695..2824 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 2925..4103 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 3156..4094 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 4051..4071 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 4110..4143 /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 4194..4227 /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 4234..4243 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4255..4311 /codon_start=1 /product="calmodulin-binding peptide from avian smooth muscle myosin light chain kinase" /label=calmodulin-binding peptide /translation="RRKWNKAGHAVRAIGRLSS" protein_bind complement(4606..4639) /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(4690..4723) /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(5323..5342) /label=mCherry-F /note="mCherry, forward primer" misc_feature 5980..6568 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6033..6053) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6451..6462) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 6600..7076 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(6799..6818) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 7116..97 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 7116..86 /label=AAV2 ITR ORIGIN 1 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg 61 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc 121 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct 181 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 241 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 301 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 361 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct 421 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 481 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt 541 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 601 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg 661 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 721 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 781 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat 841 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 901 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 961 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 1021 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 1081 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 1141 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 1201 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 1261 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 1321 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 1381 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 1441 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 1501 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 1561 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 1621 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 1681 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 1741 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 1801 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 1861 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 1921 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1981 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 2041 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 2101 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 2161 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 2221 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 2281 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2341 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2401 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2461 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2521 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2581 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2641 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca 2701 ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc 2761 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt 2821 tcctgcggcc gcacgcgtaa gctttgcaaa gatggataaa gttttaaaca gagaggaatc 2881 tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc 2941 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt 3001 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc 3061 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg 3121 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg 3181 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc 3241 agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt 3301 gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc 3361 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag 3421 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa 3481 atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg 3541 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa 3601 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt 3661 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa 3721 gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag 3781 agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt 3841 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt 3901 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg 3961 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg 4021 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt 4081 ttcttccatt tcaggtgtcg tgaggatcca taacttcgta taggatactt tatacgaagt 4141 tatgtagctg gagcaatttg aaaagattgt tgtattacct ctagctgcgc ttaataactt 4201 cgtatagtat accttatacg aagttatgtc gacgccacca tggtggactc atcacgtcgt 4261 aagtggaata aggcaggtca cgcagtcaga gctataggtc ggctgagctc acccgtggtt 4321 tccgagcgga tgtaccccga ggacggcgcc ctgaagagcg agatcaagaa ggggctgagg 4381 ctgaaggacg gcggccacta cgccgccgag gtcaagacca cctacaaggc caagaagccc 4441 gtgcagctgc ccggcgccta catcgtggac atcaagttgg acatcgtgtc ccacaacgag 4501 gactacacca tcgtggaaca gtgcgaacgc gccgagggcc gccactccac cggcggcatg 4561 gacgagctgt acaagggagg tacaggtgag tacaggaggg ctagcataac ttcgtataaa 4621 gtatcctata cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagatag 4681 gtgcaaagaa taacttcgta taaggtatac tatacgaagt tatggtaccg aagttcctat 4741 tcttcaaata gtataggaac ttcaattgtt attggtgcaa agaatcactt ccttaagtct 4801 ttatactgcc caggaagttc ctattcttca aaaggtatag gaacttcggc gcgccctact 4861 tcgctgtcat catttgtaca aactcttcgt agtttacctg accatcccca tcgatgtctg 4921 ctaccctgat catttcatca acctcctcat ctgttaactt ctctccaagg tctgtcatca 4981 cgtggcgaag ctctgctgcg ccgatgtagc cattgccgtc cttatcaaac acgcggaacg 5041 cttcgcgaat ttcctcttca ctgtctgtgt cattcatttt tcttgccatc atcgtcagga 5101 actcagggaa gtcgaatgtg ccgtcaccgt cggcatctac ttcattgatc atgtcctgca 5161 gctctgcttc tgtggggttc tgccccagcg atcggaacac cgtcccaagc tccttggttg 5221 ttatcgtccc atccccgtcc ttgtcaaata gggagaaagc ctctttaaat tctgcgatct 5281 gctcttcagt caggtcgtca cgcgtagcct cccagcccat ggtcttcttc tgcattacgg 5341 ggccgtcggg ggggaagttg gtgccgcgca gcttcacctt gtagatgaat acgccgtcct 5401 gcagggagga gtcctggttc acgtgaataa tgccgccgtc ctcgaagttc atcacgcggt 5461 cccacctgaa gccctcgggg aaggacagct tgaagtagtc ggggatgtcg gctgggtgct 5521 taatgtaggc cttggagccg tacatgaact gaggggacag gatgtcccag gcgaagggca 5581 gggggccacc cttggtcacc ttcagcttag cggtctgaaa ggcctcgtag gggcggccct 5641 cgccctcgcc ctcgatctcg aactcgtggc cgttcacgga gccctccatg tgcaccttga 5701 agcgcatgaa ctccttgatg atggccatgt tatcctcctc gcccttgctc accagactcc 5761 cgcctgtggg acgacatgac ttaaccagtt aaccatgttc tctgaacatg agaagggctg 5821 gccactctcc atctagagaa gttcctatac tatttgaaga ataggaactt cttgccttaa 5881 cccagaaatt atcactgtta ttctttagaa tagtgcaaag agaagttcct ataccttttg 5941 aagaatagga acttcgaatt cgatatcaag cttatcgata atcaacctct ggattacaaa 6001 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac 6061 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc 6121 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt 6181 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc 6241 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc 6301 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg 6361 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc cacctggatt 6421 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc 6481 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt 6541 cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga gagatctacg 6601 ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag 6661 tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct 6721 tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa 6781 cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc 6841 tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt 6901 tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg 6961 gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt 7021 ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga 7081 ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc 7141 cactccctct ctgcgcgct //