LOCUS       sequence-209146-        7242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express the genetically-encoded fluorescent ACh indicator GACh1.0 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-GACh1.0
FEATURES             Location/Qualifiers
     source          1..7242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        3..382
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        383..586
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     537..557
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     631..650
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        631..649
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     730..789
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     protein_bind    793..817
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     1878..1899
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2005..2026)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2266..2285)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2785..2814
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2818..2964
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     primer_bind     complement(2986..3003)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2992..3216
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3348..3936)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3428..3447)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4265..4312
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4337..4356
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4544..5338)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(4544..5338)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4655..4674)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5265..5284
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(5437..5456)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        5535..5726
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5683..5703
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5786..6577,6578..6646))
                     /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     6409..6428
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6647..6751)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6778..7233
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6865..6884)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7075..7096
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
       61 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      121 caacgacccc tgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      181 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      241 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      301 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      361 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      421 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      481 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      541 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
      601 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
      661 ttggtaccga gctcggatcc actagtaacg gccgccagtg tgctggaatt cggcttgggg
      721 atatccacca tggagacaga cacactcctg ctatgggtac tgctgctctg ggttccaggt
      781 tccactggtg acacaagttt gtacaaaaaa gttggcacca tgaccttgca caataacagt
      841 acaacctcgc ctttgtttcc aaacatcagc tcctcctgga tacacagccc ctccgatgca
      901 gggctgcccc cgggaaccgt cactcatttc ggcagctaca atgtttctcg agcagctggc
      961 aatttctcct ctccagacgg taccaccgat gaccctctgg gaggtcatac cgtctggcaa
     1021 gtggtcttca tcgctttctt aacgggcatc ctggccttgg tgaccatcat cggcaacatc
     1081 ctggtaattg tgtcatttaa ggtcaacaag cagctgaaga cggtcaacaa ctacttcctc
     1141 ttaagcctgg cctgtgccga tctgattatc ggggtcattt caatgaatct gtttacgacc
     1201 tacatcatca tgaatcgatg ggccttaggg aacttggcct gtgacctctg gcttgccatt
     1261 gactacgtag ccagcaatgc ctctgttatg aatcttctgg tcatcagctt tgacagatac
     1321 ttttccatca cgaggccgct cacgtaccga gccaaacgaa caacaaagag agccggtgtg
     1381 atgatcggtc tggcttgggt catctccttt gtcctttggg ctcctgccat cttgttctgg
     1441 caatactttg ttggaaagag aactgtgcct ccgggagagt gcttcattca gttcctcagt
     1501 gagcccacca ttacttttgg cacagccatc gctgcttttt atatgcctgt caccattatg
     1561 actattttat actggagggt ctttcaggag gccaaaaggc agctccagaa gattgacaaa
     1621 tctgagggcc gcttccatgt ccagaacctt agccaggtgg agcagggagg aaacgtctat
     1681 atcaaggccg acaagcagaa gaacggcatc aaggcgaact tccacatccg ccacaacatc
     1741 gaggacggcg gcgtgcagct cgcctaccac taccagcaga acacccccat cggcgacggc
     1801 cccgtgctgc tgcccgacaa ccactacctg agcgtgcagt ccaaactttc gaaagacccc
     1861 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1921 ggcatggacg agctgtacaa gggcggtacc ggagggagca tggtgagaaa gggcgaggag
     1981 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2041 ttcagcgtgt ccggcgaggg tgagggcgat gccacctacg gcaagctgac cctgaagttc
     2101 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2161 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2221 gccatgcccg aaggctacat ccaggagcgc accatcttct tcaaggacga cggcaactac
     2281 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2341 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacggagga
     2401 gcagcagcag atgggcggac ggggcatgga ctccgcagat cttccaagtt ctgcttgaag
     2461 gagcacaaag ccctcaagac gctcagtgcg atcttgcttg ccttcatcat cacttggacc
     2521 ccatacaaca tcatggttct ggtgaacacc ttttgtgaca gctgcatacc caaaaccttt
     2581 tggaatctgg gctactggct gtgctacatc aacagcaccg tgaaccccgt gtgctatgct
     2641 ctgtgcaaca aaacattcag aaccactttc aagatgctgc tgctgtgcca gtgtgacaaa
     2701 aaaaagaggc gcaagcagca gtaccagcag agacagtcgg tcatttttca caagcgcgca
     2761 cccgagcagg ccttgtaagt cgacgaacaa aaactcatct cagaagagga tctgaatgct
     2821 gtgggccagg acacgcagga ggtcatcgtg gtgccacact ccttgccctt taaggtggtg
     2881 gtgatctcag ccatcctggc cctggtggtg ctcaccatca tctcccttat catcctcatc
     2941 atgctttggc agaagaagcc acgttaggcg gccgctcgag atcagcctcg actgtgcctt
     3001 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3061 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3121 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3181 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagtg
     3241 gcggtaatac ggttatccac agaatcgggg gataacgcag gaaagaacat gtgagcaaaa
     3301 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3361 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3421 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3481 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3541 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3601 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3661 tccaacccgg taagacgcga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3721 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3781 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3841 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3901 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3961 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4021 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4081 atatatgagt aacctgaggc tatggcaggg cctgccgccc cgacgttggc tgcgagccct
     4141 gggccttcac ccgaacttgg ggggtggggt ggggaaaagg aagaaacgcg ggcgtattgg
     4201 ccccaatggg gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg aaccccgcgt
     4261 ttatgaacaa acgacccaac accgtgcgtt ttattctgtc tttttattgc cgtcatagcg
     4321 cgggttcctt ccggtattgt ctccttccgt gtttcagtta gcctccccct agggtgggcg
     4381 aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg
     4441 attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tgatggcagg
     4501 ttgggcgtcg cttggtcggt catttcgaac cccagagtcc cgctcagaag aactcgtcaa
     4561 gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga
     4621 agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt
     4681 cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat
     4741 tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt
     4801 cgggcatgct cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt
     4861 cgtccagatc 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 gccgggcctc ggcctctgca taaataaaaa
     5461 aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg ggcggaatgg
     5521 gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc
     5581 tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc
     5641 tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat
     5701 tccacagctg gttctttccg cctcaggact cttccttttt caataaatca atctaaagta
     5761 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5821 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5881 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5941 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6001 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6061 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6121 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6181 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6241 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6301 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6361 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6421 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6481 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6541 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6601 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6661 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6721 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6781 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     6841 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     6901 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     6961 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     7021 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     7081 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     7141 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     7201 cgaattttaa caaaatatta acgcttacaa tttacgcgcg cg
//
