LOCUS       sequence-236120-        6665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express the genetically-encoded fluorescent acethycholine(ACh) 
            sensor GRAB-ACh3.0 in a cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2020 Sep 28. pii: 10.1038/s41592-020-0953-2. doi: 
            10.1038/s41592-020-0953-2."
COMMENT     Sequence Label: pAAV-hSyn-DIO-GRAB_ACh3.0
FEATURES             Location/Qualifiers
     source          1..6665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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    complement(802..835)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(886..919)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     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(2775..2834)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      2831..2840
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2847..2880
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2931..2964
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     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"
     promoter        5077..5181
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5182..5250,5251..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      join(6213..6665,1..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
//
