LOCUS Exported 6665 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION express the genetically-encoded fluorescent acethycholine(ACh) sensor GRAB-ACh3.0 in a cre-dependent manner. ACCESSION . VERSION . KEYWORDS pAAV-hSyn-DIO-GRAB_ACh3.0 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6665) AUTHORS Jing M, Li Y, Zeng J, Huang P, Skirzewski M, Kljakic O, Peng W, Qian T, Tan K, Zou J, Trinh S, Wu R, Zhang S, Pan S, Hires SA, Xu M, Li H, Saksida LM, Prado VF, Bussey TJ, Prado MAM, Chen L, Cheng H, Li Y TITLE An optimized acetylcholine sensor for monitoring in vivo cholinergic activity. JOURNAL Nat Methods. 2020 Sep 28. pii: 10.1038/s41592-020-0953-2. doi: 10.1038/s41592-020-0953-2. PUBMED 32989318 REFERENCE 2 (bases 1 to 6665) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6665 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 37..56 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 198..327 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" protein_bind 802..835 /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 886..919 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 1610..1631 /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1737..1758) /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(2747..2771) /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" sig_peptide complement(2772..2834) /product="leader sequence from mouse immunoglobulin kappa light chain" /label=Ig-kappa leader regulatory 2831..2840 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(2847..2880) /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(2931..2964) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 2989..3577 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3042..3062) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3460..3471) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3609..4085 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3808..3827) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4125..4265 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4125..4254 /label=AAV2 ITR rep_origin 4340..4795 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4427..4446) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4637..4658 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4812..4831) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4931..4953 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4991..5009) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5077..5181 /gene="bla" /label=AmpR promoter CDS 5182..6042 /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(5400..5419) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6213..136 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" ORIGIN 1 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 61 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 121 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 181 gccttttgct cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg 241 ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag 301 tggccaactc catcactagg ggttcctgcg gccgcacgcg tatgaggcgg ggtgggggtg 361 cctacctgac gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg 421 cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc 481 cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc 541 tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc cttcccggcc 601 accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg aggcgcgaga 661 taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt 721 ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga aaccggtgcc 781 acctctagag tcgactccgg aataacttcg tataggatac tttatacgaa gttatgcaga 841 atggtagctg gattgtagct gctattagca atatgaaacc tcttaataac ttcgtatagc 901 atacattata cgaagttatg gcgcatatgc tacaaggcct gctcgggtgc gcgcttgtga 961 aaaatgaccg actgtctctg ctggtactgc tgcttgcgcc tctttttttt gtcacactgg 1021 cacagcagca gcatcttgaa agtggttctg aatgttttgt tgcacagagc atagcacacg 1081 gggttcacgg tgctgttgat gtagcacagc cagtagccca gattccaaaa ggttttgggt 1141 atgcagctgt cacaaaaggt gttcaccaga accatgatgt tgtatggggt ccaagtgatg 1201 atgaaggcaa gcaagatcgc actgagggtc ttggcattat ggttgtactc cagcttgtgc 1261 cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg 1321 gtgtcgccct cgaacttcac aatggcgcgg gtcttgtaat tgccgtcgcc cttgaagaag 1381 atggtgcgct cctggatgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc 1441 atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt cacgagggtg 1501 ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgatg 1561 gtggcatcgc cctcaccctc gccggtcacg ctgaacttgt ggccgtttac gtcgccgtcc 1621 agctcgacca ggatgggcac caccccggtg aacagctcct cgccctttct caccatgctc 1681 cctccggtac cgcccttgta cagctcgtcc atgccattag tgatcccggc ggcggtcacg 1741 aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttcgaaag tttggactgc 1801 acgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc 1861 tggtagtggt aggcgagctg cacgccgccg tcctcgatgt tgtggcggat gtggaagttc 1921 gccttgatgc cgttcttctg cttgtcggcc ttgatataga cgtttcctcc ttcagtttcc 1981 ttatagatcc tccagtataa aatagtcata atggtgacag gcatataaaa agcagcgatg 2041 gctgtgccaa aagtaatggt gggctcactg aggaactgaa tgaagcactc tcccggaggc 2101 acagttctct ttccaacaaa gtattgccag aacaagatgg caggagccca aaggacaaag 2161 gagatgaccc aagccagacc gatcatcaca ccggctctct ttgttgttcg tttggctcgg 2221 tacgtgagcg gcctcgtgat ggaaaagtat ctgtcaaagc tgatgaccag aagattcata 2281 acagaggcat tgctggctac gtagtcaatg gcaagccaga ggtcacaggc caagttccct 2341 aaggcccatc gattcatgat gatgtaggtc gtaaacagat tcattgaaat gaccccgata 2401 atcagatcgg cacaggccag gcttaagagg aagtagttgt tgactgtctt cagctgcttg 2461 ttgaccttaa atgacacaat taccaggatg ttgccgatga tggtcaccaa ggccaggatg 2521 cccgttaaga aagcgatgaa gaccacttgc cagacggtat gacctcccag agggtcatcg 2581 gtggtaccgt ctggagagga gaaattgcca gctgctcgag aaacattgta gctgccgaaa 2641 tgagtgacgg ttcccggggg cagccctgca tcggaggggc tgtgtatcca ggaggagctg 2701 atgtttggaa acaaaggcga ggttgtactg ttattgtgca aggtggtgcc aacttttttg 2761 tacaaacttg tgtcaccagt ggaacctgga acccagagca gcagtaccca tagcaggagt 2821 gtgtctgtct ccatggtggc gctagcataa cttcgtataa agtatcctat acgaagttat 2881 ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt 2941 ataatgtatg ctatacgaag ttatgaattc gatatcaagc ttatcgataa tcaacctctg 3001 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta 3061 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt 3121 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc 3181 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt 3241 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg 3301 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac 3361 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc 3421 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac 3481 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct 3541 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag 3601 agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg 3661 ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact 3721 aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg 3781 ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac 3841 aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc 3901 ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt 3961 agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct 4021 acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg 4081 tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat 4141 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt 4201 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct 4261 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 4321 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 4381 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc 4441 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc 4501 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt 4561 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 4621 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg 4681 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag 4741 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg 4801 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca 4861 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct 4921 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg 4981 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt 5041 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 5101 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 5161 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 5221 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 5281 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 5341 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 5401 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 5461 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 5521 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 5581 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 5641 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 5701 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 5761 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 5821 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 5881 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 5941 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 6001 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 6061 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 6121 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 6181 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 6241 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 6301 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt 6361 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 6421 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 6481 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 6541 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 6601 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 6661 ggcag //