LOCUS Exported 5933 bp ds-DNA circular SYN 05-JAN-2023 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-jGCaMP7f-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5933) 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 5933) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 5, 2023 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5933 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(24..471) /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" repeat_region 553..682 /label=AAV2 ITR rep_origin 754..1209 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(841..860) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1051..1072 /label=F1ori-F /note="F1 origin, forward primer" promoter 1235..1339 /gene="bla" /label=AmpR promoter CDS 1340..2200 /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(1558..1577) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2371..2959 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2860..2879 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3113..3130 /label=L4440 /note="L4440 vector, forward primer" repeat_region 3196..3325 /label=AAV2 ITR polyA_signal 3487..3608 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3524..3543) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3578..3597 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" misc_feature 3651..4239 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" CDS 3757..3768 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind 4166..4186 /label=WPRE-R /note="WPRE, reverse primer" protein_bind complement(4281..4314) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(4385..4418) /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)." regulatory 4429..4438 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4444..4461 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 4465..4497 /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10" /label=T7 tag (gene 10 leader) /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS 4501..4524 /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /label=Xpress(TM) tag /translation="DLYDDDDK" regulatory 4531..4540 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4552..4608 /codon_start=1 /product="calmodulin-binding peptide from avian smooth muscle myosin light chain kinase" /label=calmodulin-binding peptide /translation="RRKWNKTGHAVRAIGRLSS" primer_bind 4821..4842 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(4948..4969) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5209..5228) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" protein_bind 5798..5831 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 5894..5927 /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)." ORIGIN 1 ccttgctagc agcttgaatt cgactgcgct ctcaggcacg acacgactcc tccgctgccc 61 accgcagact gaggcagcgc tgagtcgccg gcgccgcagc gcagatggtc gcgcccgtgc 121 ccccctatct cgcgcctcgc gtggtgcggt ccggctgggc cggcggcggc gcggacgcga 181 ccaaggtggc cgggaagggg agtttgcggg ggaccggcga gtgacgtcag cgcgccttca 241 gtgctgaggc ggcggtggcg cgcgccgcca ggcgggggcg aaggcactgt ccgcggtgct 301 gaagctggca gtgcgcacgc gcctcgccgc atcctgtttc ccctccccct ctctgatagg 361 ggatgcgcaa tttggggaat gggggttggg tgcttgtcca gtgggtcggg gtcggtcgtc 421 aggtaggcac ccccaccccg cctcatcctg gtcctaaaac ccacttgcac tcatacgcag 481 ggccctctgc agagatcttc ctagagcatg gctacgtaga taagtagcat ggcgggttaa 541 tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 601 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 661 cagtgagcga gcgagcgcgc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 721 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg 781 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 841 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 901 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 961 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 1021 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 1081 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 1141 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 1201 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 1261 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 1321 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 1381 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 1441 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 1501 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 1561 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 1621 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 1681 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 1741 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 1801 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 1861 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 1921 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 1981 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 2041 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 2101 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 2161 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 2221 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 2281 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 2341 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 2401 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 2461 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct 2521 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 2581 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 2641 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 2701 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 2761 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 2821 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 2881 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 2941 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 3001 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 3061 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 3121 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc 3181 gattcattaa tgcagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg 3241 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg 3301 gccaactcca tcactagggg ttccttgtag ttaatgatta acccgccatg ctacttatct 3361 acgtagccat gctctaggaa gatccttatc gattttacca catttgtaga ggttttactt 3421 gctttaaaaa acctcccaca tctccccctg aacctgaaac ataaaatgaa tgcaattgtt 3481 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 3541 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 3601 gtatcttatc atgtctgctc gaagcggccg cccgggtcga cggtatcgat gcggggaggc 3661 ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa gaggccgcag 3721 agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag ggacgtagca 3781 gaaggacgtc ccgcgcagaa tccaggtggc aacacaggcg agcagccaag gaaaggacga 3841 tgatttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc ctgtccagca 3901 gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga aagcgaaagt 3961 cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg cgtcagcaaa 4021 cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat aaagagacag 4081 caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa tagcatgata 4141 caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt taagaatacc 4201 agtcaatctt tcacaaattt tgtaatccag aggttgatta tcgataagct tgatatcgaa 4261 ttcgaacctc ttcgagggac ataacttcgt ataatgtatg ctatacgaag ttatgggtcg 4321 atggtgagat ctggactaga gggtcgatgg tgatgcttgg atcaaaatag gaagaatccc 4381 aatgataact tcgtataaag tatcctatac gaagttatac taggatccgc caccatgggt 4441 tctcatcatc atcatcatca tggtatggct agcatgactg gtggacagca aatgggtcgg 4501 gatctgtacg acgatgacga taaggatctc gccaccatgg tcgactcatc acgtcgtaag 4561 tggaataaga caggtcacgc agtcagagct ataggtcggc tgagctcact cgagaacgtc 4621 tatatcaagg ccgacaagca gaagaacggc atcaaggcga acttcaagat ccgccacaac 4681 atcgaggacg gcggcgtgca gctcgcctac cactaccagc agaacacccc catcggcgac 4741 ggccccgtgc tgctgcccga caaccactac ctgagcgtgc agtccaaact ttcgaaagac 4801 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 4861 ctcggcatgg acgagctgta caagggcggt accggaggga gcatggtgag caagggcgag 4921 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 4981 aagttcagcg tgtccggcga gggtgagggc gatgccacct acggcaagct gaccctgaag 5041 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc 5101 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag 5161 tccgccatgc ccgaaggcta catccaggag cgcaccatct tcttcaagga cgacggcaac 5221 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctt 5281 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacctg 5341 ccggaccaac tgactgaaga gcagatcgca gaatttaaag agctgttctc cctatttgac 5401 aaggacgggg atgggacaat aacaaccaag gagctgggga cggtgatgcg gtctctgggg 5461 cagaacccca cagaagcaga gctgcaggac atgatcaatg aagtagatgc cgacggtgac 5521 ggcacaatcg acttccctga gttcctgaca atgatggcaa gaaaaatgaa atacacagac 5581 agtgaagaag aaattagaga agcgttccgt gtgtttgata aggatggcaa tggctacatc 5641 agtgcagcag agcttcgcca cgtgatgaca aaccttggag agaagttaac agatgaagag 5701 gttgatgaaa tgatcaggga agcagacatc gatggggatg gtcaggtaaa ctacgaagag 5761 tttgtacaaa tgatgacagc gaagtgaaag ctctagtata acttcgtata gcatacatta 5821 tacgaagtta tgggtcgatg gtgatgcttg gcaattcggg tcgatggtga agcattggtc 5881 ttcctatttt gatataactt cgtataggat actttatacg aagttatggg gat //