LOCUS Exported 6272 bp ds-DNA linear SYN 20-MAY-2021 DEFINITION AAV expression of Cre recombinase-activated ultrasensitive protein calcium sensor from CAG promoter. ACCESSION . VERSION . KEYWORDS pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6272) AUTHORS Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS TITLE Ultrasensitive fluorescent proteins for imaging neuronal activity. JOURNAL Nature. 2013 Jul 18;499(7458):295-300. doi: 10.1038/nature12354. PUBMED 23868258 REFERENCE 2 (bases 1 to 6272) AUTHORS . TITLE Direct Submission JOURNAL Exported May 20, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6272 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 367..389 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 451..470 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind complement(505..538) /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(601..634) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(645..1997) /codon_start=1 /product="improved fluorescent protein-based calcium sensor (Chen et al., 2013)" /label=GCaMP6m /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKGSYRDTEEEIREAFGVFDKDGN GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" primer_bind 1204..1223 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1463..1484 /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1590..1611) /label=EGFP-C /note="EGFP, forward primer" regulatory 1892..1901 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 1994..2003 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 2014..2047 /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 2118..2151 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 2193..2781 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2246..2266) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2664..2675) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 2824..2945 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2835..2854) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2889..2908 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 3107..3236 /label=AAV2 ITR primer_bind complement(3302..3319) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3473..4061) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3553..3572) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4232..5092) /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 4855..4874 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5093..5197) /gene="bla" /label=AmpR promoter rep_origin complement(5223..5678) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5360..5381) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5572..5591 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 5750..5879 /label=AAV2 ITR ORIGIN 1 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc 61 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc 121 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca 181 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg 241 gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg 301 cggggggagc ggctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca 361 gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc 421 atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc 481 attttggcaa agaattggat ccccataact tcgtataaag tatcctatac gaagttatat 541 caaaatagga agaccaatgc ttcaccatcg acccgaattg ccaagcatca ccatcgaccc 601 ataacttcgt ataatgtatg ctatacgaag ttatactaga gctttcactt cgctgtcatc 661 atttgtacaa actcttcgta gtttacctga ccatccccat cgatgtctgc ttccctgatc 721 atttcatcaa cctcttcatc tgttaacttc tctccaaggt ttgtcatcac gtggcgaagc 781 tctgctgcac tgatgtagcc attgccatcc ttatcaaaca caccgaacgc ttctctaatt 841 tcttcttccg tgtccctgta gctccctttt cttgccatca ttgtcaggaa ctcagggaag 901 tcgattgtgc cgtcaccgtc ggcatctact tcattgatca tgtcctgcag ctctgcttct 961 gtggggttct gccccagaga ccgcatcacc gtccccagct ccttggttgt tattgtccca 1021 tccccgtcct tgtcaaatag ggagaaagcc tctttaaatt ctgcgatctg ctcttcagtc 1081 agttggtccg gcaggttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag 1141 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg 1201 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggat gtagccttcg 1261 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac 1321 tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg 1381 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcacc ctcgccggac 1441 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg 1501 gtgaacagct cctcgccctt gctcaccatg ctccctccgg taccgccctt gtacagctcg 1561 tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc 1621 ttctcgttgg ggtctttcga aagtttggac tgcacgctca ggtagtggtt gtcgggcagc 1681 agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtaggcgag ctgcacgccg 1741 ccgtcctcga tgttgtggcg gatcttgaag ttcgccttga tgccgttctt ctgcttgtcg 1801 gccttgatat agacgttctc gagtgagctc agccgaccta tagctctgac tgcgtgacct 1861 gtcttattcc acttacgacg tgatgagtcg accatggtgg cgagatcctt atcgtcatcg 1921 tcgtacagat cccgacccat ttgctgtcca ccagtcatgc tagccatacc atgatgatga 1981 tgatgatgag aacccatggt ggcggatcct agtataactt cgtataggat actttatacg 2041 aagttatcat tgggattctt cctattttga tccaagcatc accatcgacc ctctagtcca 2101 gatctcacca tcgacccata acttcgtata gcatacatta tacgaagtta tgtccctcga 2161 agaggttcga attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg 2221 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 2281 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 2341 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 2401 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 2461 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 2521 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 2581 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg 2641 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 2701 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct 2761 ccctttgggc cgcctccccg catcgatacc gtcgacccgg gcggccgctt cgagcagaca 2821 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 2881 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 2941 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg 3001 ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cgataaggat cttcctagag 3061 catggctacg tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg 3121 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 3181 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc 3241 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 3301 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 3361 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 3421 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 3481 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 3541 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 3601 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 3661 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 3721 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 3781 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 3841 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 3901 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 3961 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 4021 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 4081 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 4141 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 4201 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 4261 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 4321 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 4381 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 4441 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 4501 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 4561 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 4621 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 4681 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 4741 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 4801 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 4861 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 4921 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 4981 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 5041 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 5101 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 5161 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 5221 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 5281 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 5341 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 5401 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 5461 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 5521 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 5581 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 5641 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 5701 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tgcgcgctcg 5761 ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc 5821 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctt 5881 gtagttaatg attaacccgc catgctactt atctacgtag ccatgctcta ggaagagtac 5941 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 6001 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 6061 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 6121 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 6181 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 6241 cacccccaat tttgtattta tttatttttt aa //