LOCUS Exported 7169 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 7169) 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 7169) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7169 /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)" CDS 1571..1627 /codon_start=1 /product="calmodulin-binding peptide from avian smooth muscle myosin light chain kinase" /label=calmodulin-binding peptide /translation="RRKWNKAGHAVRAIGRLSS" protein_bind complement(1922..1955) /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(2006..2039) /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(2639..2658) /label=mCherry-F /note="mCherry, forward primer" misc_feature 3296..3884 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3349..3369) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3767..3778) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3916..4392 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4115..4134) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4432..4572 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4432..4561 /label=AAV2 ITR rep_origin 4647..5102 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4734..4753) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4944..4965 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5119..5138) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5238..5260 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5298..5316) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5384..5488 /gene="bla" /label=AmpR promoter CDS 5489..6349 /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(5707..5726) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6520..7108 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7009..7028 /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 tatgtcgacg ccaccatggt 1561 ggactcatca cgtcgtaagt ggaataaggc aggtcacgca gtcagagcta taggtcggct 1621 gagctcaccc gtggtttccg agcggatgta ccccgaggac ggcgccctga agagcgagat 1681 caagaagggg ctgaggctga aggacggcgg ccactacgcc gccgaggtca agaccaccta 1741 caaggccaag aagcccgtgc agctgcccgg cgcctacatc gtggacatca agttggacat 1801 cgtgtcccac aacgaggact acaccatcgt ggaacagtgc gaacgcgccg agggccgcca 1861 ctccaccggc ggcatggacg agctgtacaa gggaggtaca ggtgagtaca ggagggctag 1921 cataacttcg tataaagtat cctatacgaa gttatttgcc ttaacccaga aattatcact 1981 gttattcttt agataggtgc aaagaataac ttcgtataag gtatactata cgaagttatg 2041 gtaccgaagt tcctattctt caaatagtat aggaacttca attgttattg gtgcaaagaa 2101 tcacttcctt aagtctttat actgcccagg aagttcctat tcttcaaaag gtataggaac 2161 ttcggcgcgc cctacttcgc tgtcatcatt tgtacaaact cttcgtagtt tacctgacca 2221 tccccatcga tgtctgctac cctgatcatt tcatcaacct cctcatctgt taacttctct 2281 ccaaggtctg tcatcacgtg gcgaagctct gctgcgccga tgtagccatt gccgtcctta 2341 tcaaacacgc ggaacgcttc gcgaatttcc tcttcactgt ctgtgtcatt catttttctt 2401 gccatcatcg tcaggaactc agggaagtcg aatgtgccgt caccgtcggc atctacttca 2461 ttgatcatgt cctgcagctc tgcttctgtg gggttctgcc ccagcgatcg gaacaccgtc 2521 ccaagctcct tggttgttat cgtcccatcc ccgtccttgt caaataggga gaaagcctct 2581 ttaaattctg cgatctgctc ttcagtcagg tcgtcacgcg tagcctccca gcccatggtc 2641 ttcttctgca ttacggggcc gtcggggggg aagttggtgc cgcgcagctt caccttgtag 2701 atgaatacgc cgtcctgcag ggaggagtcc tggttcacgt gaataatgcc gccgtcctcg 2761 aagttcatca cgcggtccca cctgaagccc tcggggaagg acagcttgaa gtagtcgggg 2821 atgtcggctg ggtgcttaat gtaggccttg gagccgtaca tgaactgagg ggacaggatg 2881 tcccaggcga agggcagggg gccacccttg gtcaccttca gcttagcggt ctgaaaggcc 2941 tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc 3001 tccatgtgca ccttgaagcg catgaactcc ttgatgatgg ccatgttatc ctcctcgccc 3061 ttgctcacca gactcccgcc tgtgggacga catgacttaa ccagttaacc atgttctctg 3121 aacatgagaa gggctggcca ctctccatct agagaagttc ctatactatt tgaagaatag 3181 gaacttcttg ccttaaccca gaaattatca ctgttattct ttagaatagt gcaaagagaa 3241 gttcctatac cttttgaaga ataggaactt cgaattcgat atcaagctta tcgataatca 3301 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt 3361 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc 3421 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc 3481 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg 3541 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc 3601 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 3661 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta 3721 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc 3781 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct 3841 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct 3901 gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg 3961 gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg 4021 tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg 4081 caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca 4141 gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct 4201 cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt 4261 ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag 4321 gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc 4381 ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc 4441 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 4501 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 4561 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 4621 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 4681 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 4741 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 4801 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 4861 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 4921 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 4981 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 5041 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 5101 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 5161 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 5221 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 5281 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 5341 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 5401 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 5461 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 5521 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 5581 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 5641 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 5701 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 5761 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 5821 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 5881 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 5941 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 6001 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 6061 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 6121 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 6181 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 6241 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 6301 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 6361 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 6421 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 6481 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 6541 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 6601 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 6661 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 6721 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 6781 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 6841 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 6901 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 6961 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 7021 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 7081 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 7141 ggccttttgc tggccttttg ctcacatgt //