LOCUS Exported 6605 bp ds-DNA linear SYN 31-MAY-2021 DEFINITION AAV expression of FLEXed Twitch2B driven by CAG promoter. Twitch 2B is a fluorescent reporter for calcium signaling. ACCESSION . VERSION . KEYWORDS pAAV.CAG.Flex.Twitch2B.WPRE.SV40 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6605) AUTHORS Thestrup T, Litzlbauer J, Bartholomaus I, Mues M, Russo L, Dana H, Kovalchuk Y, Liang Y, Kalamakis G, Laukat Y, Becker S, Witte G, Geiger A, Allen T, Rome LC, Chen TW, Kim DS, Garaschuk O, Griesinger C, Griesbeck O TITLE Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. JOURNAL Nat Methods. 2014 Jan 5. doi: 10.1038/nmeth.2773. PUBMED 24390440 REFERENCE 2 (bases 1 to 6605) AUTHORS . TITLE Direct Submission JOURNAL Exported May 31, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6605 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 406..428 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 490..509 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind 543..576 /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 627..660 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1669..1690) /label=EGFP-C /note="EGFP, forward primer" primer_bind 2018..2037 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2277..2298 /label=EGFP-N /note="EGFP, reverse primer" regulatory 2340..2349 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(2362..2395) /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(2446..2479) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 2492..3080 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2545..2565) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2963..2974) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3123..3244 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3134..3153) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3188..3207 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 3406..3535 /label=AAV2 ITR primer_bind complement(3601..3618) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3772..4360) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3852..3871) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4531..5391) /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 5154..5173 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5392..5496) /gene="bla" /label=AmpR promoter rep_origin complement(5522..5977) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5659..5680) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5871..5890 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 6049..6178 /label=AAV2 ITR ORIGIN 1 ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg 61 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg 121 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg 181 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 241 ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct 301 gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcggggct 361 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc 421 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac 481 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc 541 ttataacttc gtataggata ctttatacga agttatgcag aatggtagct ggattgtagc 601 tgctattagc aatatgaaac ctcttaataa cttcgtatag catacattat acgaagttat 661 actagttgat catcaatcct caatgttgtg acggatctta aaattagcct taatgccgtt 721 cttttgctta tcggctgtga tgtaaacatt gtgactgttg tagttgtatt ccaacttatg 781 cccgagtatg tttccgtcct ccttaaagtc gattcccttc agctcgattc ggttgacaag 841 tgtatcccct tcgaatttca cctcggccct agtcttgtaa ttaccatcgt ccttgaagaa 901 gatagttctc tcctggacgt acccctctgg catggctgat ttaaagaaat cgtgttgttt 961 catatggtct gggtagcggg cgaaacacat aaggccatat cctagagtag taacgagtgt 1021 gggccatggc acgggcagtt tccccgttgt gcagatgagc ttgagtgtca atttgccatt 1081 agttgcgtcc ccctcaccct cccctcgcac gctgaattta tgaccgttga catccccatc 1141 cagttccacc aaaataggga ctacaccagt gaacaattct tcgcccttgc tcaccataga 1201 tcccccggtc ccacctttgt acagctcgtc cattccgaga gtgattccgg cggcggtgac 1261 aaattccagc aggaccatgt gatcccgctt ttcattagga tctttggaaa gcttggactg 1321 gtatgacagg tagtggttgt caggcaggag aaccggccca tccccgatcg gagtattctg 1381 ctggtagtga tctgcaagct ggaccccacc catgagctcg gggtagatgg gctgcacgtt 1441 ttccaccatc ttcaggaact cgtcgaaatc gattctgccg ttcttgtcgg tgtcggagtc 1501 gccgaagatc tcgtccacgt cctcgtcggt cagctgctcg ccggtcagtc tgatgatgtc 1561 gccaaactcc tctctgtcga tgaagccgtt gccgtcgaag tcgaagattc tgaagcactc 1621 gctcagttcc tcttcgctgg cgtcggcaac ttgcatgcgg gcggcggtca cgaactccag 1681 caggaccatg tgatcgcgct tctcgttggg gtctttgctc agcttggact gggtgctcag 1741 gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg 1801 gtcggcgagc tgcacgctgc cgtcctcgat gttgcagttg aggccgaagt tggccttgat 1861 gccgttcttc tgcttgtcgg cggtgatata gacgttgccg tggatggcgt tgtactccag 1921 cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt 1981 caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt 2041 gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg 2101 ctgcttcatg tggtcggggt agcgggcgaa gcactgcacg ccccagctca gggtggtcac 2161 gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagctt 2221 gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc 2281 gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac 2341 catggtggcg ctagcctcga gataacttcg tataaagtat cctatacgaa gttatttgcc 2401 ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac ttcgtataat 2461 gtatgctata cgaagttata agcttatcga taatcaacct ctggattaca aaatttgtga 2521 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 2581 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 2641 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt 2701 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct 2761 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg 2821 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 2881 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg 2941 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct 3001 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc 3061 cctttgggcc gcctccccgc atcgataccg tcgacccggg cggccgcttc gagcagacat 3121 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 3181 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 3241 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt 3301 tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc ttcctagagc 3361 atggctacgt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga 3421 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg 3481 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca 3541 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 3601 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 3661 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 3721 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 3781 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 3841 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 3901 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 3961 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 4021 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 4081 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 4141 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 4201 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 4261 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 4321 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 4381 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 4441 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 4501 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 4561 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 4621 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 4681 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 4741 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 4801 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 4861 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 4921 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 4981 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 5041 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 5101 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 5161 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 5221 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 5281 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 5341 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 5401 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 5461 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 5521 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca 5581 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag 5641 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc 5701 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc 5761 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc 5821 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa 5881 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc 5941 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc 6001 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct gcgcgctcgc 6061 tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc 6121 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttccttg 6181 tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag gaagagtacc 6241 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 6301 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 6361 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 6421 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 6481 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 6541 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 6601 ggggg //