LOCUS       sequence-200841-        7242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express the genetically-encoded fluorescent ACh indicator GACh2.0 in
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7242)
  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 7242)
  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: pDisplay-GACh2.0
FEATURES             Location/Qualifiers
     source          1..7242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        10..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     544..564
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     638..657
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        638..656
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     737..796
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     protein_bind    800..824
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     1885..1906
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2012..2033)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2273..2292)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2792..2821
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2992..3009)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2998..3222
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3354..3942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3434..3453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4271..4318
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4343..4362
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4550..5338)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDQEHQGL
                     APAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIALA
                     TRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4661..4680)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5265..5284
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5373..5733)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5387..5525
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5542..5733
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5690..5710
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5793..6584,6585..6653))
                     /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     6416..6435
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6654..6758)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6785..7240
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6872..6891)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7082..7103
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
      601 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
      661 cccaagcttg gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgg
      721 cttggggata tccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt
      781 tccaggttcc actggtgaca caagtttgta caaaaaagtt ggcaccatga ccttgcacaa
      841 taacagtaca acctcgcctt tgtttccaaa catcagctcc tcctggatac acagcccctc
      901 cgatgcaggg ctgcccccgg gaaccgtcac tcatttcggc agctacaatg tttctcgagc
      961 agctggcaat ttctcctctc cagacggtac caccgatgac cctctgggag gtcataccgt
     1021 ctggcaagtg gtcttcatcg ctttcttaac gggcatcctg gccttggtga ccatcatcgg
     1081 caacatcctg gtaattgtgt catttaaggt caacaagcag ctgaagacgg tcaacaacta
     1141 cttcctctta agcctggcct gtgccgatct gattatcggg gtcatttcaa tgaatctgtt
     1201 tacgacctac atcatcatga atcgatgggc cttagggaac ttggcctgtg acctctggct
     1261 tgccattgac tacgtagcca gcaatgcctc tgttatgaat cttctggtca tcagctttga
     1321 cagatacttt tccatcacga ggccgctcac gtaccgagcc aaacgaacaa caaagagagc
     1381 cggtgtgatg atcggtctgg cttgggtcat ctcctttgtc ctttgggctc ctgccatctt
     1441 gttctggcaa tactttgttg gaaagagaac tgtgcctccg ggagagtgct tcattcagtt
     1501 cctcagtgag cccaccatta cttttggcac agccatcgct gctttttata tgcctgtcac
     1561 cattatgact attttatact ggagggtctt tcaggaggcc aaaaggcagc tccagaagat
     1621 tgacaaatct gagggccgct tccatgtcca gaaccttagc caggtggagc agggaggaaa
     1681 cgtctatatc aaggccgaca agcagaagaa cggcatcaag gcgaacttcc acatccgcca
     1741 caacatcgag gacggcggcg tgcagctcgc ctaccactac cagcagaaca cccccatcgg
     1801 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc gtgcagtcca aactttcgaa
     1861 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1921 cactctcggc atggacgagc tgtacaaggg cggtaccgga gggagcatgg tgagaaaggg
     1981 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     2041 ccacaagttc agcgtgtccg gcgagggtga gggcgatgcc acctacggca agctgaccct
     2101 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     2161 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     2221 caagtccgcc atgcccgaag gctacatcca ggagcgcacc atcttcttca aggacgacgg
     2281 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     2341 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     2401 cgctcctagc gtcgcagatg ggcggacggg gcatggactc cgcagatctt ccaagttctg
     2461 cttgaaggag cacaaagccc tcaagacgct cagtgcgatc ttgcttgcct tcatcatcac
     2521 ttggacccca tacaacatca tggttctggt gaacaccttt tgtgacagct gcatacccaa
     2581 aaccttttgg aatctgggct actggctgtg ctacatcaac agcaccgtga accccgtgtg
     2641 ctatgctctg tgcaacaaaa cattcagaac cactttcaag atgctgctgc tgtgccagtg
     2701 tgacaaaaaa aagaggcgca agcagcagta ccagcagaga cagtcggtca tttttcacaa
     2761 gcgcgcaccc gagcaggcct tgtaagtcga cgaacaaaaa ctcatctcag aagaggatct
     2821 gaatgctgtg ggccaggaca cgcaggaggt catcgtggtg ccacactcct tgccctttaa
     2881 ggtggtggtg atctcagcca tcctggccct ggtggtgctc accatcatct cccttatcat
     2941 cctcatcatg cttggcagaa gaagccacgt taggcggccg ctcgagatca gcctcgactg
     3001 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3061 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3121 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3181 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3241 ccagtggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3301 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3361 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3421 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3481 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3541 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3601 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3661 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3721 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3781 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3841 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3901 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3961 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4021 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4081 taaagtatat atgagtaacc tgaggctatg gcagggcctg ccgccccgac gttggctgcg
     4141 agccctgggc cttcacccga acttgggggg tggggtgggg aaaaggaaga aacgcgggcg
     4201 tattggcccc aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc
     4261 ccgcgtttat gaacaaacga cccaacaccg tgcgttttat tctgtctttt tattgccgtc
     4321 atagcgcggg ttccttccgg tattgtctcc ttccgtgttt cagttagcct ccccctaggg
     4381 tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg
     4441 aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat
     4501 ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact
     4561 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     4621 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     4681 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     4741 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
     4801 cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     4861 gctcttgatc atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc
     4921 gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca
     4981 ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct
     5041 gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca
     5101 cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca
     5161 gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg
     5221 acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga
     5281 atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc
     5341 gaaacgatcc tcatcctgtc tcttgatcga tctttgcaaa agcctaggcc tccaaaaaag
     5401 cctcctcact acttctggaa tagctcagag gccgaggagg cggcctcggc ctctgcataa
     5461 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     5521 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     5581 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     5641 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     5701 cacacattcc acagctggtt ctttccgcct caggactctt cctttttcaa taaatcaatc
     5761 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5821 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5881 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5941 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6001 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6061 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6121 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6181 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6241 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6301 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6361 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6421 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6481 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6541 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6601 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6661 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6721 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6781 cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     6841 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     6901 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     6961 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     7021 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     7081 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     7141 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     7201 tttaacgcga attttaacaa aatattaacg cttacaattt ac
//
