LOCUS       sequence-232154-        6544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express the genetically-encoded fluorescent acethycholine(ACh) 
            sensor GACh2.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6544)
  AUTHORS   Jing M, Zhang P, Wang G, Feng J, Mesik L, Zeng J, Jiang H, Wang S, 
            Looby JC, Guagliardo NA, Langma LW, Lu J, Zuo Y, Talmage DA, Role 
            LW, Barrett PQ, Zhang LI, Luo M, Song Y, Zhu JJ, Li Y
  TITLE     A genetically encoded fluorescent acetylcholine indicator for in 
            vitro and in vivo studies.
  JOURNAL   Nat Biotechnol. 2018 Sep;36(8):726-737. doi: 10.1038/nbt.4184. Epub 
            2018 Jul 9.
  PUBMED    29985477
REFERENCE   2  (bases 1 to 6544)
  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"; doi: "10.1038/nbt.4184"; 
            journalName: "Nat Biotechnol"; date: "2018-09"; volume: "36"; issue:
            "8"; pages: "726-737"
COMMENT     Sequence Label: pAAV-hSyn-GACh2.0
FEATURES             Location/Qualifiers
     source          1..6544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     regulatory      592..601
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     598..657
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     protein_bind    661..685
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     1746..1767
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1873..1894)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2134..2153)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2671..3259
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2724..2744)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3142..3153)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3291..3767
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3490..3509)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3807..3947
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3807..3936
                     /label=AAV2 ITR
     rep_origin      4022..4477
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4109..4128)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4319..4340
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4494..4513)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4613..4635
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4759..4863
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4864..4932,4933..5724)
                     /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(5082..5101)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5895..6483
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6384..6403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtatgag gcggggtggg ggtgcctacc
      181 tgacgaccga ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc
      241 ccctatcaga gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt
      301 cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca
      361 ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg
      421 gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg
      481 ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg
      541 tgggcagcgg aggagtcgtg tcgtgcctga gagcgcagtc gagaaaccgg tgccaccatg
      601 gagacagaca cactcctgct atgggtactg ctgctctggg ttccaggttc cactggtgac
      661 acaagtttgt acaaaaaagt tggcaccatg accttgcaca ataacagtac aacctcgcct
      721 ttgtttccaa acatcagctc ctcctggata cacagcccct ccgatgcagg gctgcccccg
      781 ggaaccgtca ctcatttcgg cagctacaat gtttctcgag cagctggcaa tttctcctct
      841 ccagacggta ccaccgatga ccctctggga ggtcataccg tctggcaagt ggtcttcatc
      901 gctttcttaa cgggcatcct ggccttggtg accatcatcg gcaacatcct ggtaattgtg
      961 tcatttaagg tcaacaagca gctgaagacg gtcaacaact acttcctctt aagcctggcc
     1021 tgtgccgatc tgattatcgg ggtcatttca atgaatctgt ttacgaccta catcatcatg
     1081 aatcgatggg ccttagggaa cttggcctgt gacctctggc ttgccattga ctacgtagcc
     1141 agcaatgcct ctgttatgaa tcttctggtc atcagctttg acagatactt ttccatcacg
     1201 aggccgctca cgtaccgagc caaacgaaca acaaagagag ccggtgtgat gatcggtctg
     1261 gcttgggtca tctcctttgt cctttgggct cctgccatct tgttctggca atactttgtt
     1321 ggaaagagaa ctgtgcctcc gggagagtgc ttcattcagt tcctcagtga gcccaccatt
     1381 acttttggca cagccatcgc tgctttttat atgcctgtca ccattatgac tattttatac
     1441 tggagggtct ttcaggaggc caaaaggcag ctccagaaga ttgacaaatc tgagggccgc
     1501 ttccatgtcc agaaccttag ccaggtggag cagggaggaa acgtctatat caaggccgac
     1561 aagcagaaga acggcatcaa ggcgaacttc cacatccgcc acaacatcga ggacggcggc
     1621 gtgcagctcg cctaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1681 cccgacaacc actacctgag cgtgcagtcc aaactttcga aagaccccaa cgagaagcgc
     1741 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1801 ctgtacaagg gcggtaccgg agggagcatg gtgagaaagg gcgaggagct gttcaccggg
     1861 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1921 ggcgagggtg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1981 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     2041 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2101 ggctacatcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2161 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2221 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acgctcctag cgtcgcagat
     2281 gggcggacgg ggcatggact ccgcagatct tccaagttct gcttgaagga gcacaaagcc
     2341 ctcaagacgc tcagtgcgat cttgcttgcc ttcatcatca cttggacccc atacaacatc
     2401 atggttctgg tgaacacctt ttgtgacagc tgcataccca aaaccttttg gaatctgggc
     2461 tactggctgt gctacatcaa cagcaccgtg aaccccgtgt gctatgctct gtgcaacaaa
     2521 acattcagaa ccactttcaa gatgctgctg ctgtgccagt gtgacaaaaa aaagaggcgc
     2581 aagcagcagt accagcagag acagtcggtc atttttcaca agcgcgcacc cgagcaggcc
     2641 ttgtaagaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     2701 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     2761 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     2821 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     2881 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     2941 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3001 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3061 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     3121 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3181 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     3241 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc
     3301 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     3361 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     3421 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     3481 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     3541 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     3601 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     3661 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     3721 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     3781 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     3841 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     3901 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     3961 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     4021 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4081 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4141 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4201 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     4261 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4321 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta
     4381 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4441 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     4501 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     4561 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4621 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     4681 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4741 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4801 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4861 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4921 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4981 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5041 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5101 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5161 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5221 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5281 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5341 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5401 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     5461 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5521 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     5581 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     5641 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5701 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     5761 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     5821 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5881 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5941 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6001 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     6061 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6121 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6181 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6241 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6301 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6361 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6421 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6481 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     6541 atgt
//
