LOCUS       sequence-236118-        6389 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express the genetically-encoded fluorescent acethycholine(ACh) 
            sensor GRAB-ACh3.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6389)
  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 6389)
  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-GRAB_ACh3.0
FEATURES             Location/Qualifiers
     source          1..6389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      553..562
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     559..618
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     protein_bind    622..646
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     1635..1656
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1762..1783)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    2488..3076
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2541..2561)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2959..2970)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3108..3584
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3307..3326)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3624..3764
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3624..3753
                     /label=AAV2 ITR
     rep_origin      3839..4294
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3926..3945)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4136..4157
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4311..4330)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4430..4452
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4576..4680
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4681..4749,4750..5541)
                     /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(4899..4918)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5712..6300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6201..6220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6362..6389,1..102)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
       61 cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc acgcgtatga
      121 ggcggggtgg gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc
      181 ccattcccca aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag
      241 gcgcgtgcgc actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg
      301 cgcgccaccg ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa
      361 ctccccttcc cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc
      421 acgcgaggcg cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact
      481 cagcgctgcc tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcgcagt
      541 cgagaaaccg gtgccaccat ggagacagac acactcctgc tatgggtact gctgctctgg
      601 gttccaggtt ccactggtga cacaagtttg tacaaaaaag ttggcaccac cttgcacaat
      661 aacagtacaa cctcgccttt gtttccaaac atcagctcct cctggataca cagcccctcc
      721 gatgcagggc tgcccccggg aaccgtcact catttcggca gctacaatgt ttctcgagca
      781 gctggcaatt tctcctctcc agacggtacc accgatgacc ctctgggagg tcataccgtc
      841 tggcaagtgg tcttcatcgc tttcttaacg ggcatcctgg ccttggtgac catcatcggc
      901 aacatcctgg taattgtgtc atttaaggtc aacaagcagc tgaagacagt caacaactac
      961 ttcctcttaa gcctggcctg tgccgatctg attatcgggg tcatttcaat gaatctgttt
     1021 acgacctaca tcatcatgaa tcgatgggcc ttagggaact tggcctgtga cctctggctt
     1081 gccattgact acgtagccag caatgcctct gttatgaatc ttctggtcat cagctttgac
     1141 agatactttt ccatcacgag gccgctcacg taccgagcca aacgaacaac aaagagagcc
     1201 ggtgtgatga tcggtctggc ttgggtcatc tcctttgtcc tttgggctcc tgccatcttg
     1261 ttctggcaat actttgttgg aaagagaact gtgcctccgg gagagtgctt cattcagttc
     1321 ctcagtgagc ccaccattac ttttggcaca gccatcgctg ctttttatat gcctgtcacc
     1381 attatgacta ttttatactg gaggatctat aaggaaactg aaggaggaaa cgtctatatc
     1441 aaggccgaca agcagaagaa cggcatcaag gcgaacttcc acatccgcca caacatcgag
     1501 gacggcggcg tgcagctcgc ctaccactac cagcagaaca cccccatcgg cgacggcccc
     1561 gtgctgctgc ccgacaacca ctacctgagc gtgcagtcca aactttcgaa agaccccaac
     1621 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactaatggc
     1681 atggacgagc tgtacaaggg cggtaccgga gggagcatgg tgagaaaggg cgaggagctg
     1741 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1801 agcgtgaccg gcgagggtga gggcgatgcc accatcggca agctgaccct gaagttcatc
     1861 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     1921 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1981 atgcccgaag gctacatcca ggagcgcacc atcttcttca agggcgacgg caattacaag
     2041 acccgcgcca ttgtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2101 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ccataatgcc
     2161 aagaccctca gtgcgatctt gcttgccttc atcatcactt ggaccccata caacatcatg
     2221 gttctggtga acaccttttg tgacagctgc atacccaaaa ccttttggaa tctgggctac
     2281 tggctgtgct acatcaacag caccgtgaac cccgtgtgct atgctctgtg caacaaaaca
     2341 ttcagaacca ctttcaagat gctgctgctg tgccagtgtg acaaaaaaaa gaggcgcaag
     2401 cagcagtacc agcagagaca gtcggtcatt tttcacaagc gcgcacccga gcaggccttg
     2461 taggaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
     2521 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     2581 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     2641 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     2701 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     2761 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     2821 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     2881 aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg
     2941 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     3001 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     3061 tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct
     3121 gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc
     3181 ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta
     3241 tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct
     3301 gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct
     3361 ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg
     3421 catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat
     3481 tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat
     3541 tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa
     3601 ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     3661 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     3721 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     3781 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     3841 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3901 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3961 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4021 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     4081 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4141 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     4201 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4261 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     4321 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4381 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4441 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     4501 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     4561 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4621 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4681 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4741 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4801 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4861 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4921 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     4981 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5041 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5101 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5161 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5221 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5281 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5341 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5401 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5461 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5521 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5581 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5641 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5701 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5761 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5821 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5881 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5941 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6001 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6061 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6121 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6181 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6241 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6301 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6361 tcctgcaggc agctgcgcgc tcgctcgct
//
