LOCUS Exported 7028 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Intersectional viral expression of ic++-EYFP in cells expressing Cre AND NOT Flp. ACCESSION . VERSION . KEYWORDS pAAV-nEF-Con/Foff 2.0-iC++-EYFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7028) 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 7028) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7028 /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 188..399 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 412..680 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" protein_bind 708..741 /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)" regulatory 834..843 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 1371..1404 /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 1455..1488 /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)." primer_bind 1601..1622 /label=EGFP-N /note="EGFP, reverse primer" protein_bind complement(2254..2287) /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 complement(2338..2371) /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)." primer_bind complement(2593..2612) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" CDS 2752..3003 /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 2940..2961 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(3013..3046) /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 3155..3743 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3208..3228) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3626..3637) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3775..4251 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3974..3993) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4291..4431 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4291..4420 /label=AAV2 ITR rep_origin 4506..4961 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4593..4612) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4803..4824 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4978..4997) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5097..5119 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5157..5175) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5243..5347 /gene="bla" /label=AmpR promoter CDS 5348..6208 /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(5566..5585) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6379..6967 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6868..6887 /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 cgcgtaagga tctgcgatcg ctccggtgcc 181 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc 241 aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac 301 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg 361 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg aggggctcgc 421 atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg gttgagtcgc 481 gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta 541 aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc tagactcagc 601 cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt ttcgttttct 661 gttctgcgcc gttacagatc caagctgtga ccggcgccta cggatccgaa gttcctattc 721 tctagaaagt ataggaactt cgcagagtag tagctggatt gtagctgcta ttagcaatct 781 aaaacctctt agaagttcct attcttcaaa aggtatagga acttcggcgc gccgccacca 841 tggactatgg cggcgctttg tctgccgtcg gactcttcca aacctcatac actcttgaga 901 acaatggttc tgttatttgc atcccgaaca acggccagtg cttctgcttg gcttggctta 961 aatccaacgg aacaaatgcc gagaagttgg ctgccaacat tctgcagtgg atttcttttg 1021 cgctttcagc gctctgcctg atgttctacg gctaccagac ctggaagtct acttgcggct 1081 gggagaatat ttacgtggcc acgatccaga tgatcaagtt catcatcgag tatttccata 1141 gctttgacga acctgcggtg atctactcat ccaacggcaa caagaccagg tggcttcgtt 1201 acgcgagctg gctgctgacc tgccctgtca ttcttatcca cctgagcaac cttacgggtc 1261 tggcgaacga ctataacaag cgtaccatgg gtctgctggt gtcagatatc ggcacgatcg 1321 tgtggggcac cacggccgcg ctgtccaagg taagtgtcgg ggttgagctc ataacttcgt 1381 atagtatacc ttatacgaag ttattctttg caccattcta aagaataaca gtgataattt 1441 ctgggttaag gcaaataact tcgtatagga tactttatac gaagttatgg tacccctcct 1501 gtactcacct tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg 1561 ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc 1621 aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccacgtt gatgtcgatc 1681 tggtccaggg ggatgtactc gccctcgctg gtgatcctgc tcttggcggc cgcgaccgcg 1741 ccagcctcgg cctcgtcctc caccagcgtc tcgacctcaa tctcagtgcc accaatgttc 1801 aatttggtgg tcttgcgaat gtcgccgtgg atgaggatat ggctgtggat cagcacgcgc 1861 aggtagtggc cgagcagacc ccagcactgc ttcgacatca ggtcaatgat ggtgtggccg 1921 acgttggagc cgtacctgct caggacgccg aagccctcgg ggccgaggat gaacaggatg 1981 gggaacatac cccatgatac gaagaagagc caagccatgc cagtcaccac ctggcgacac 2041 cggcccttcg gcacggtgtg gtacgcctca atgtagacct tggctgcgtt gaagaatgtg 2101 tagatgccgt agcacaggcc catcaggaag aaaatgacac ggacgtatcc ctaaacacat 2161 gagaaataga gtcataagca catgggtcac atacaggaga tatgtatata acattaatac 2221 aattttataa aaaaaagggg gggcacaact agtataactt cgtataaggt atactatacg 2281 aagttattaa gaggtttcat attgctaata gcagctacaa tccagctacc attctgcata 2341 acttcgtata aagtatccta tacgaagtta tgtcgactgg agagtggcca gcccttctca 2401 tgttcagaga acatggttaa ctggttaagt catgtcgtcc cacagctgcc cgtgccctgg 2461 cccaccctcg tgaccacctt cggctacggc ctgcagtgct tcgcccgcta ccccgaccac 2521 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 2581 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 2641 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 2701 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 2761 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 2821 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 2881 aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 2941 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 3001 aagtaagcta gcgaagttcc tatactttct agagaatagg aacttctaag cagacaatct 3061 tgtaatacat cagcagctag tagattgctc cagctagaag ttcctatacc ttttgaagaa 3121 taggaacttc gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg 3181 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc 3241 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta 3301 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt 3361 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca 3421 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc 3481 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt 3541 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg 3601 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg 3661 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat 3721 ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagagat ctacgggtgg 3781 catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc 3841 accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat 3901 aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt 3961 agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact 4021 gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga 4081 ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc 4141 accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct 4201 cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg 4261 taggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc 4321 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 4381 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga 4441 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc 4501 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 4561 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 4621 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 4681 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag 4741 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 4801 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga 4861 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 4921 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac 4981 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 5041 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 5101 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct 5161 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg 5221 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 5281 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 5341 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 5401 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 5461 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 5521 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 5581 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 5641 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 5701 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 5761 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 5821 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 5881 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 5941 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 6001 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 6061 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 6121 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 6181 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 6241 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 6301 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 6361 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 6421 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 6481 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 6541 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 6601 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 6661 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 6721 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 6781 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 6841 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 6901 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 6961 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 7021 tcacatgt //