LOCUS Exported 6986 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses the Fluorescence Lifetime Imaging Microscopy (FLIM)-compatible PKA activity sensor FLIM-AKAR in a Cre dependent manner in an AAV backbone. ACCESSION . VERSION . KEYWORDS AAV-FLEX-FLIM-AKAR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6986) AUTHORS Chen Y, Saulnier JL, Yellen G, Sabatini BL TITLE A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging. JOURNAL Front Pharmacol. 2014 Apr 2;5:56. doi: 10.3389/fphar.2014.00056. eCollection 2014. PUBMED 24765076 REFERENCE 2 (bases 1 to 6986) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6986 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(11..28) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(182..770) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(262..281) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(941..1801) /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 1564..1583 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1802..1906) /gene="bla" /label=AmpR promoter rep_origin complement(1932..2387) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2069..2090) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2281..2300 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 2459..2588 /label=AAV2 ITR promoter 2901..3178 /label=chicken beta-actin promoter primer_bind 3458..3480 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 3542..3561 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind complement(3596..3629) /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(3692..3725) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(5164..5682) /codon_start=1 /product="N-terminal fragment of mVenus for use in bimolecular fluorescence complementation (BiFC) (Kodama and Hu, 2010)" /label=VN173 /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIE" primer_bind 5357..5376 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5616..5637 /label=EGFP-N /note="EGFP, reverse primer" regulatory 5679..5688 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 5709..5742 /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 5813..5846 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 5888..6476 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5941..5961) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6359..6370) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 6519..6640 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6530..6549) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 6584..6603 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 6802..6931 /label=AAV2 ITR ORIGIN 1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt 2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 2641 gaagatcgta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 2701 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 2761 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 2821 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 2881 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc 2941 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat 3001 gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg 3061 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc 3121 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg 3181 agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc 3241 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 3301 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa 3361 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg 3421 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc 3481 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg 3541 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga tccccataac 3601 ttcgtataaa gtatcctata cgaagttata tcaaaatagg aagaccaatg cttcaccatc 3661 gacccgaatt gccaagcatc accatcgacc cataacttcg tataatgtat gctatacgaa 3721 gttatactag ggatcccctt actcgatgtt gtgcctgatt ttgaagttga ccttgatacc 3781 gtttttctgc ttgtcggcca taatatagac gttcacgcta ttccagttgt actccagctt 3841 atgcccgagg atatttccat cttctttaaa gtcaatcccc ttcagctcga ttcggttgac 3901 gagggtgtca ccttcgaact tcacctctgc gcgagttttg tagttgccat cgtccttaaa 3961 gaaaattgtc cgttcctgca cgtatccctc aggcatggca gatttaaaga aatcatgctg 4021 cttcatgtgg tcggggtacc ttgcaaagca catcaggccg taaccgaagg tagtgaccag 4081 ggttggccaa ggcacgggca gcttccctgt ggtgcagatc agtttgagtg tcagctttcc 4141 ataggtagca tccccttctc cctcgccgct cacgctgaac ttgtggccgt tcacgtcgcc 4201 gtccagttcc acgagaatgg ggaccacgcc agtgaacagt tcctcgccct tggacaccat 4261 gctaccccct gttccgcctt tgtacagctc gtccatcccg agagtgattc cagcggctgt 4321 gactctttcg agcagcacca tatgatccct tttctcattt ggatcttttg acagggcact 4381 ctgataagag aggtagtggt tgtctgggag gaggactggc ccgtcaccga ttggagtatt 4441 ctgctgatag tggtctgcga gctggactga acccatgagt tctgagccgc cggtgccgcc 4501 gtccaccagg gtggcgcgac gcaggccctt ggtgctgccc tcgccgctgc ctggcttacc 4561 tgcagagcga tcaactttgt tctgctcgag gcactgctta aatttgtcgt ttatgaaaat 4621 gaccagagat aaaatatctg attccacgcc tacaccaacg gttatttcat caccttgaga 4681 cagtaactga ttgctgttct tctcgacttt ttgcccattt aaccaggtcc cgttagtgct 4741 gatgtcgttg aggaggaggt tcccgtcttc gcccaggaga atctgaaagt gcttattgga 4801 cagcctgcta atgttcccca ggtgatagtc gcaagcgggg tttcttccga atgtccagac 4861 tttcttaata gaccttttct ccttcagcac ctgactgatg tcggcagaca ggtcgcgaat 4921 ggggatctgt ccagttgtac agatgacccg gcacacgata ttttcaccga tctgctcctg 4981 ggaaaactta tgcatgcgag cggcagtgac gaactcgagc agcaccatgt gatcccgttt 5041 ctcgtttggg tcttttgaga gcttactctg agtagacaga taatgattat cagggagcag 5101 gacgggcccg tctccgatag gtgtgttctg ctggtaatga tcagccagct gcacgctgcc 5161 gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc 5221 catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc 5281 gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa 5341 cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg 5401 gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta 5461 gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg 5521 cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc 5581 gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat 5641 gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggcgg cgggtaccga 5701 attctagtat aacttcgtat aggatacttt atacgaagtt atcattggga ttcttcctat 5761 tttgatccaa gcatcaccat cgaccctcta gtccagatct caccatcgac ccataacttc 5821 gtatagcata cattatacga agttatgtcc ctcgaagagg ttcgaattcg atatcaagct 5881 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta 5941 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc 6001 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga 6061 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac 6121 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc 6181 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc 6241 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg 6301 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc 6361 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc 6421 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg 6481 ataccgtcga cccgggcggc cgcttcgagc agacatgata agatacattg atgagtttgg 6541 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 6601 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca 6661 ttttatgttt caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta 6721 caaatgtggt aaaatcgata aggatcttcc tagagcatgg ctacgtagat aagtagcatg 6781 gcgggttaat cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc 6841 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc 6901 gggcggcctc agtgagcgag cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg 6961 gagaggcggt ttgcgtattg ggcgct 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