LOCUS Exported 6379 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV-mediated expression of ultrasensitive protein calcium sensor under the CAG promoter, Cre mediated flip-excision switch. ACCESSION . VERSION . KEYWORDS pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6379) AUTHORS Dana H, Sun Y, Mohar B, Hulse BK, Kerlin AM, Hasseman JP, Tsegaye G, Tsang A, Wong A, Patel R, Macklin JJ, Chen Y, Konnerth A, Jayaraman V, Looger LL, Schreiter ER, Svoboda K, Kim DS TITLE High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. JOURNAL Nat Methods. 2019 Jul;16(7):649-657. doi: 10.1038/s41592-019-0435-6. Epub 2019 Jun 17. PUBMED 31209382 REFERENCE 2 (bases 1 to 6379) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6379 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR promoter 441..718 /label=chicken beta-actin promoter primer_bind 997..1019 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1081..1100 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind complement(1135..1168) /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(1231..1264) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(1275..2627) /codon_start=1 /product="improved fluorescent protein-based calcium sensor (Chen et al., 2013)" /label=GCaMP6s /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK TGHAVRVIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRS LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" primer_bind 1834..1853 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2093..2114 /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2220..2241) /label=EGFP-C /note="EGFP, forward primer" regulatory 2522..2531 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 2624..2633 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 2644..2677 /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 2748..2781 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 2823..3411 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2876..2896) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3294..3305) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3454..3575 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3465..3484) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3519..3538 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 3737..3866 /label=AAV2 ITR primer_bind complement(3932..3949) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4103..4691) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4183..4202) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4862..5722) /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 5485..5504 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5723..5827) /gene="bla" /label=AmpR promoter rep_origin complement(5853..6308) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5990..6011) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 6202..6221 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 181 aggaagagta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 421 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc 481 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat 541 gggggcgggg gggggggggg ggcgcgcgcc aggcggggsg gggsggggcg aggggcgggg 601 cggsgcgwgg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc 661 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg 721 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc 781 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 841 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag 901 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg 961 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg 1021 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg 1081 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattggat ccccataact 1141 tcgtataaag tatcctatac gaagttatat caaaatagga agaccaatgc ttcaccatcg 1201 acccgaattg ccaagcatca ccatcgaccc ataacttcgt ataatgtatg ctatacgaag 1261 ttatactaga gctttcactt cgctgtcatc atttgtacaa actcttcgta gtttacctga 1321 ccatccccat cgatgtctgc ttccctgatc atttcatcaa cctcttcatc tgttaacttc 1381 tctccaaggt ttgtcatcac gtggcgaagc tctgctgcac tgatgtagcc attgccatcc 1441 ttatcaaaca caccgaacgc ttctctaatt tcttcttccg tgtccctgta tttcattttt 1501 cttgccatca ttgtcaggaa ctcagggaag tcgattgtgc cgtcaccgtc ggcatctact 1561 tcattgatca tgtcctgcag ctctgcttct gtggggttct gccccagaga ccgcatcacc 1621 gtccccagct ccttggttgt tattgtccca tccccgtcct tgtcaaatag ggagaaaagc 1681 tctttaaatt ctgcgatctg ctcttcagtc agttggtcag gaaggttgta ctccagcttg 1741 tgccccagga tgttgccgtc ctccttgaag tcgatgccct taagctcgat gcggttcacc 1801 agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag 1861 aagatggtgc gctcctggat gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc 1921 ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg 1981 gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg 2041 taggtggcat cgccctcacc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg 2101 tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg 2161 ctccctccgg taccgccctt gtacagctcg tccatgccga gagtgatccc ggcggcggtc 2221 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttcga aagtttggac 2281 tgcacgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc 2341 tgctggtagt ggtaggcgag ctgcacgccg ccgtcctcga tgttgtggcg gatgtggaag 2401 ttcgccttga tgccgttctt ctgcttgtcg gccttgatat agacgttctc gagtgagctc 2461 agccgaccta tcactctgac tgcgtgacct gtcttattcc acttacgacg tgatgagtcg 2521 accatggtgg cgagatcctt atcgtcatcg tcgtacagat cccgacccat ttgctgtcca 2581 ccagtcatgc tagccatacc atgatgatga tgatgatgag aacccatggt ggcggatcct 2641 agtataactt cgtataggat actttatacg aagttatcat tgggattctt cctattttga 2701 tccaagcatc accatcgacc ctctagtcca gatctcacca tcgacccata acttcgtata 2761 gcatacatta tacgaagtta tgtccctcga agaggttcga attcgatatc aagcttatcg 2821 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg 2881 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc 2941 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt 3001 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca 3061 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc 3121 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc 3181 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc 3241 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc 3301 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc 3361 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc 3421 gtcgacccgg gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa 3481 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt 3541 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta 3601 tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat 3661 gtggtaaaat cgataaggat cttcctagag catggctacg tagataagta gcatggcggg 3721 ttaatcatta actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct 3781 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg 3841 gcctcagtga gcgagcgagc gcgcagctgc attaatgaat cggccaacgc gcggggagag 3901 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 3961 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 4021 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 4081 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 4141 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 4201 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 4261 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 4321 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 4381 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 4441 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 4501 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 4561 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 4621 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 4681 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 4741 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 4801 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 4861 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 4921 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 4981 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 5041 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 5101 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 5161 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 5221 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 5281 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 5341 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 5401 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 5461 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 5521 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 5581 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 5641 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 5701 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 5761 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 5821 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa 5881 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa 5941 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa 6001 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca 6061 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg 6121 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc 6181 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc 6241 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca 6301 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 6361 ctcttcgcta ttacgccag //