LOCUS       Exported                6598 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Mammalian cell expression plasmid for red fluorescent Ca2+ indicator
            K-GECO1.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA-K-GECO1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6598)
  AUTHORS   Shen Y, Dana H, Abdelfattah AS, Patel R, Shea J, Molina RS, Rawal B,
            Rancic V, Chang YF, Wu L, Chen Y, Qian Y, Wiens MD, Hambleton N, 
            Ballanyi K, Hughes TE, Drobizhev M, Kim DS, Koyama M, Schreiter ER, 
            Campbell RE
  TITLE     A genetically encoded Ca(2+) indicator based on circularly 
            permutated sea anemone red fluorescent protein eqFP578.
  JOURNAL   BMC Biol. 2018 Jan 16;16(1):9. doi: 10.1186/s12915-018-0480-0.
  PUBMED    29338710
REFERENCE   2  (bases 1 to 6598)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC Biol.";
            date: "2018-01-16"; volume: "16(1):9. doi"; pages: " 
            10.1186/s12915-018-0480-0"
FEATURES             Location/Qualifiers
     source          1..6598
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        490..693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     644..664
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     738..757
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        738..756
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      781..790
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     complement(2064..2081)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2070..2294
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2340..2768
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2427..2446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2637..2658
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2777..2797)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2782..3111
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2962..3097
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3024..3043
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3178..3972
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3232..3251)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3842..3861
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4146..4267
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4183..4202)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4237..4256
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4316..4332)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4316..4332)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4329..4351)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4340..4356
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4364..4394)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4409..4430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4547..4564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4718..5306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4798..4817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5477..6337)
                     /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     6100..6119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6338..6442)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6517..6536)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"