LOCUS Exported 7255 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Forebrain principle neuron specific expression of GCaMP6s Ca sensor and physically separate nuclear localized dTomato fluorophore . ACCESSION . VERSION . KEYWORDS AAV-CaMKIIa-GCaMP6s-P2A-nls-dTomato SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7255) TITLE Ting GCaMP6 plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 7255) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7255 /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 164..1452 /label=CaMKII promoter /note="promoter for the mouse calcium/calmodulin-dependent kinase II alpha subunit" regulatory 1465..1474 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1471..2820 /codon_start=1 /product="improved fluorescent protein-based calcium sensor (Chen et al., 2013)" /label=GCaMP6s /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" regulatory 1567..1576 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 1857..1878 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1984..2005) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2245..2264) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" CDS 2836..2892 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 2905..2925 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2926..3624 /codon_start=1 /product="dimeric variant of DsRed fluorescent protein (Shaner et al., 2004)" /label=dTomato /note="mammalian codon-optimized" /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH LFLYGMDELYK" primer_bind complement(3056..3074) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 3324..3343 /label=mCherry-F /note="mCherry, forward primer" primer_bind 3518..3541 /label=DsRed1-C /note="DsRed1, forward primer" misc_feature 3674..4262 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3727..3747) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4145..4156) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 4295..4406 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 4518..4658 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4518..4647 /label=AAV2 ITR rep_origin 4733..5188 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4820..4839) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5030..5051 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5205..5224) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5324..5346 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5384..5402) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5470..5574 /gene="bla" /label=AmpR promoter CDS 5575..6435 /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(5793..5812) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6606..7194 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7095..7114 /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 cgcgtttaat taacattatg gccttaggtc 181 acttcatctc catggggttc ttcttctgat tttctagaaa atgagatggg ggtgcagaga 241 gcttcctcag tgacctgccc agggtcacat cagaaatgtc agagctagaa cttgaactca 301 gattactaat cttaaattcc atgccttggg ggcatgcaag tacgatatac agaaggagtg 361 aactcattag ggcagatgac caatgagttt aggaaagaag agtccagggc agggtacatc 421 tacaccaccc gcccagccct gggtgagtcc agccacgttc acctcattat agttgcctct 481 ctccagtcct accttgacgg gaagcacaag cagaaactgg gacaggagcc ccaggagacc 541 aaatcttcat ggtccctctg ggaggatggg tggggagagc tgtggcagag gcctcaggag 601 gggccctgct gctcagtggt gacagatagg ggtgagaaag cagacagagt cattccgtca 661 gcattctggg tctgtttggt acttcttctc acgctaaggt ggcggtgtga tatgcacaat 721 ggctaaaaag cagggagagc tggaaagaaa caaggacaga gacagaggcc aagtcaacca 781 gaccaattcc cagaggaagc aaagaaacca ttacagagac tacaaggggg aagggaagga 841 gagatgaatt agcttcccct gtaaacctta gaacccagct gttgccaggg caacggggca 901 atacctgtct cttcagagga gatgaagttg ccagggtaac tacatcctgt ctttctcaag 961 gaccatccca gaatgtggca cccactagcc gttaccatag caactgcctc tttgccccac 1021 ttaatcccat cccgtctgtt aaaagggccc tatagttgga ggtgggggag gtaggaagag 1081 cgatgatcac ttgtggacta agtttgttcg catccccttc tccaaccccc tcagtacatc 1141 accctggggg aacagggtcc acttgctcct gggcccacac agtcctgcag tattgtgtat 1201 ataaggccag ggcaaagagg agcaggtttt aaagtgaaag gcaggcaggt gttggggagg 1261 cagttaccgg ggcaacggga acagggcgtt tcggaggtgg ttgccatggg gacctggatg 1321 ctgacgaagg ctcgcgaggc tgtgagcagc cacagtgccc tgctcagaag ccccaagctc 1381 gtcagtcaag ccggttctcc gtttgcactc aggagcacgg gcaggcgagt ggcccctagt 1441 tctgggggca gcgggggatc cggcgccacc atgggttctc atcatcatca tcatcatggt 1501 atggctagca tgactggtgg acagcaaatg ggtcgggatc tgtacgacga tgacgataag 1561 gatctcgcca ccatggtcga ctcatcacgt cgtaagtgga ataagacagg tcacgcagtc 1621 agagctatag gtcggctgag ctcactcgag aacgtctata tcaaggccga caagcagaag 1681 aacggcatca aggcgaactt ccacatccgc cacaacatcg aggacggcgg cgtgcagctc 1741 gcctaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 1801 cactacctga gcgtgcagtc caaactttcg aaagacccca acgagaagcg cgatcacatg 1861 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 1921 ggcggtaccg gagggagcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 1981 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt 2041 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 2101 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 2161 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc 2221 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 2281 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 2341 ggcaacatcc tggggcacaa gctggagtac aacctgccgg accaactgac tgaagagcag 2401 atcgcagaat ttaaagaggc tttctcccta tttgacaagg acggggatgg gacaataaca 2461 accaaggagc tggggacggt gatgcggtct ctggggcaga accccacaga agcagagctg 2521 caggacatga tcaatgaagt agatgccgac ggtgacggca caatcgactt ccctgagttc 2581 ctgacaatga tggcaagaaa aatgaaatac agggacacgg aagaagaaat tagagaagcg 2641 ttcggtgtgt ttgataagga tggcaatggc tacatcagtg cagcagagct tcgccacgtg 2701 atgacaaacc ttggagagaa gttaacagat gaagaggttg atgaaatgat cagggaagca 2761 gacatcgatg gggatggtca ggtaaactac gaagagtttg tacaaatgat gacagcgaag 2821 gataacggaa gcggagccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag 2881 aatcccgggc catctagaat ggcccccaag aagaagagga aggtggtgag caagggcgag 2941 gaggtcatca aagagttcat gcgcttcaag gtgcgcatgg agggctccat gaacggccac 3001 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag 3061 ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc 3121 atgtacggct ccaaggcgta cgtgaagcac cccgccgaca tccccgatta caagaagctg 3181 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggtctggtg 3241 accgtgaccc aggactcctc cctgcaggac ggcacgctga tctacaaggt gaagatgcgc 3301 ggcaccaact tcccccccga cggccccgta atgcagaaga agaccatggg ctgggaggcc 3361 tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca ccaggccctg 3421 aagctgaagg acggcggcca ctacctggtg gagttcaaga ccatctacat ggccaagaag 3481 cccgtgcaac tgcccggcta ctactacgtg gacaccaagc tggacatcac ctcccacaac 3541 gaggactaca ccatcgtgga acagtacgag cgctccgagg gccgccacca cctgttcctg 3601 tacggcatgg acgagctgta caagtaagtc gacggcgcgc ccctgcaggg aattcgatat 3661 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct 3721 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 3781 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct 3841 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga 3901 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc 3961 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac 4021 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt 4081 tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt 4141 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc 4201 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc 4261 gcatcgatac cgagcgctgc tcgagagatc tcgactgtgc cttctagttg ccagccatct 4321 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 4381 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 4441 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgcacgt 4501 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc 4561 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 4621 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct 4681 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc 4741 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 4801 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 4861 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 4921 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc 4981 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 5041 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt 5101 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 5161 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat 5221 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 5281 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 5341 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 5401 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 5461 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 5521 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 5581 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 5641 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 5701 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 5761 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 5821 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 5881 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 5941 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 6001 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 6061 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 6121 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 6181 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 6241 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 6301 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 6361 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 6421 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 6481 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 6541 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 6601 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 6661 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 6721 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 6781 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6841 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6901 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6961 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 7021 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 7081 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 7141 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 7201 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt //