LOCUS Exported 5788 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV-mediated expression of ultrasensitive protein calcium sensor under the Syn promoter, Cre mediated flip-excision switch. ACCESSION . VERSION . KEYWORDS pGP-AAV-syn-FLEX-jGCaMP7b-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5788) 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 5788) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5788 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 45..174 /label=AAV2 ITR promoter 256..703 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" protein_bind complement(733..766) /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(829..862) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(873..2225) /codon_start=1 /product="improved fluorescent protein-based calcium sensor (Chen et al., 2013)" /label=GCaMP6f /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGNILGHKLEYNPPDQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRS LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMYARKMKYRDTEEEIREAFGVFDKDGN GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" primer_bind 1432..1451 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1691..1712 /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1818..1839) /label=EGFP-C /note="EGFP, forward primer" regulatory 2120..2129 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 2222..2231 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 2242..2275 /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 2346..2379 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 2421..3009 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2474..2494) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2892..2903) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3012..3028) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3013..3029) /label=pBluescriptKS /note="For pBluescript vector" polyA_signal 3058..3179 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3069..3088) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3123..3142 /label=SV40pA-R /note="SV40 polyA, reverse primer" rep_origin complement(3556..4144) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3636..3655) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4315..5175) /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 4938..4957 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5176..5280) /gene="bla" /label=AmpR promoter rep_origin complement(5306..5761) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5443..5464) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5655..5674 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctgcgc gctcgctcgc 61 tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag 121 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt 181 taatgattaa cccgccatgc tacttatcta cgtagccatg ctctaggaag atctctgcag 241 agggccctgc gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc 301 ctacctgacg accgaccccg acccactgga caagcaccca acccccattc cccaaattgc 361 gcatccccta tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc 421 agcttcagca ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct 481 cagcactgaa ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca 541 ccttggtcgc gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat 601 aggggggcac gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc 661 tgcggtgggc agcggaggag tcgtgtcgtg cctgagagcg cagtcgaatt caagctgcta 721 gcaaggatcc ccataacttc gtataaagta tcctatacga agttatatca aaataggaag 781 accaatgctt caccatcgac ccgaattgcc aagcatcacc atcgacccat aacttcgtat 841 aatgtatgct atacgaagtt atactagagc tttcacttcg ctgtcatcat ttgtacaaac 901 tcttcgtagt ttacctgacc atccccatcg atgtctgctt ccctgatcat ttcatcaacc 961 tcttcatctg ttaacttctc tccaaggttt gtcatcacgt ggcgaagctc tgctgcactg 1021 atgtagccat tgccatcctt atcaaacaca ccgaacgctt ctctaatttc ttcttccgtg 1081 tccctgtatt tcatttttct tgcgtacatt gtcaggaact cagggaagtc gattgtgccg 1141 tcaccgtcgg catctacttc attgatcatg tcctgcagct ctgcttctgt ggggttctgc 1201 cccagagacc gcatcaccgt ccccagctcc ttggttgtta ttgtcccatc cccgtccttg 1261 tcaaataggg agaaaagctc tttaaattct gcgatctgct cttcagtcag ttggtcaggt 1321 gggttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgccctta 1381 agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag 1441 ttgccgtcgt ccttgaagaa gatggtgcgc tcctggatgt agccttcggg catggcggac 1501 ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg cacgccgtag 1561 gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac 1621 ttcagggtca gcttgccgta ggtggcatcg ccctcaccct cgccggacac gctgaacttg 1681 tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc 1741 tcgcccttgc tcaccatgct ccctccggta ccgcccttgt acagctcgtc catgccgaga 1801 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg 1861 tctttcgaaa gtttggactg cacgctcagg tagtggttgt cgggcagcag cacggggccg 1921 tcgccgatgg gggtgttctg ctggtagtgg taggcgagct gcacgccgcc gtcctcgatg 1981 ttgtggcgga tcttgaagtt cgccttgatg ccgttcttct gcttgtcggc cttgatatag 2041 acgttctcga gtgagctcag ccgacctata gctctgactg cgtgacctgt cttattccac 2101 ttacgacgtg atgagtcgac catggtggcg agatccttat cgtcatcgtc gtacagatcc 2161 cgacccattt gctgtccacc agtcatgcta gccataccat gatgatgatg atgatgagaa 2221 cccatggtgg cggatcctag tataacttcg tataggatac tttatacgaa gttatcattg 2281 ggattcttcc tattttgatc caagcatcac catcgaccct ctagtccaga tctcaccatc 2341 gacccataac ttcgtatagc atacattata cgaagttatg tccctcgaag aggttcgaat 2401 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg 2461 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt 2521 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc 2581 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt 2641 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga 2701 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct 2761 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat 2821 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct 2881 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc 2941 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg 3001 cctccccgca tcgataccgt cgacctcgac ccgggcggcc gcttcgagca gacatgataa 3061 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt 3121 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta 3181 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt 3241 aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatcttcct agagcatggc 3301 tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag 3361 ttggccactc cctctctgcg cgctcgctcr ctsactcgct gcgctcggtc gttcggctgn 3421 cggcgagcgg tatcagctca ctncaaaggc ggtaatacgg ttatccacag aatcagggga 3481 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 3541 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 3601 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 3661 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 3721 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 3781 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 3841 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 3901 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 3961 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 4021 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 4081 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 4141 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 4201 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 4261 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 4321 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 4381 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 4441 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 4501 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 4561 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 4621 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 4681 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 4741 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 4801 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 4861 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 4921 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 4981 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 5041 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 5101 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 5161 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 5221 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 5281 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg 5341 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct 5401 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt 5461 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat 5521 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca 5581 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac 5641 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta 5701 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg 5761 tcccattcgc cattcaggct gcgcaact //