LOCUS       40924_14715        7242 bp DNA     circular SYN 13-MAY-2021
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)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7242)
  AUTHORS   .
  TITLE     Direct Submission
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     SGRef: number: 2; type: "Journal Article"
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"
     promoter        638..656
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     737..799
                     /product="leader sequence from mouse immunoglobulin kappa
                     light chain"
                     /label=Ig-kappa leader
     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"
     CDS             1697..1948
                     /codon_start=1
                     /label=VC155
                     /note="C-terminal fragment of mVenus for use in bimolecular
                     fluorescence complementation (BiFC) (Kodama and Hu, 2010)"
                     /translation="DKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNH
                     YLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     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
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
                     /translation="EQKLISEEDL"
     CDS             2825..2971
                     /codon_start=1
                     /label=PDGFR-beta TM domain
                     /note="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     polyA_signal    2999..3223
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3355..3943)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    complement(4272..4319)
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4344..4363
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4554..5345)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(5380..5733)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             complement(5796..6653)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6654..6758)
                     /label=AmpR promoter
     rep_origin      6785..7240
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccctgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggagtattt 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 ctttggcaga agaagccacg ttaggcggcc gctcgagatc agcctcgact
     3001 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     3061 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     3121 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     3181 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     3241 accagtggcg gtaatacggt tatccacaga atcgggggat aacgcaggaa agaacatgtg
     3301 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3361 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3421 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3481 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3541 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3601 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3661 tcttgagtcc aacccggtaa gacgcgactt atcgccactg gcagcagcca ctggtaacag
     3721 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3781 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3841 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3901 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3961 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4021 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4081 ctaaagtata tatgagtaac ctgaggctat ggcagggcct gccgccccga cgttggctgc
     4141 gagccctggg ccttcacccg aacttggggg gtggggtggg gaaaaggaag aaacgcgggc
     4201 gtattggccc caatggggtc tcggtggggt atcgacagag tgccagccct gggaccgaac
     4261 cccgcgttta tgaacaaacg acccaacacc gtgcgtttta ttctgtcttt ttattgccgt
     4321 catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tccccctagg
     4381 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg
     4441 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga
     4501 tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac
     4561 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc
     4621 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac
     4681 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag
     4741 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc
     4801 tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga
     4861 tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc
     4921 tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc
     4981 cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg
     5041 agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg
     5101 tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg
     5161 tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc
     5221 tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca
     5281 tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca
     5341 atcatgcgaa acgatcctca tcctgtctct tgatcgatct ttgcaaaagc ctaggcctcc
     5401 aaaaaagcct cctcactact tctggaatag ctcagaggcc gggcctcggc ctctgcataa
     5461 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     5521 ggaatgggcg 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
//
