LOCUS       Exported                7242 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  express the genetically-encoded fluorescent ACh indicator GACh2.0 in
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pDisplay-GACh2.0
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
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"
FEATURES             Location/Qualifiers
     source          1..7242
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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..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"
     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"
     CDS             2825..2971
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     primer_bind     complement(2993..3010)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     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"
     primer_bind     complement(3435..3454)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    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(4551..5345)
                     /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(4662..4681)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5272..5291
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(5444..5463)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5690..5710
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(5793..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=f1 ori
                     /note="f1 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"