LOCUS       Exported                6758 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    cytoplasmic EKAR (Cerulean-Venus)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6758)
  AUTHORS   Harvey CD, Ehrhardt AG, Cellurale C, Zhong H, Yasuda R, Davis RJ, 
            Svoboda K.
  TITLE     A genetically encoded fluorescent sensor of ERK activity
  JOURNAL   PNAS 2008 Dec 9;105(49):19264-9
  PUBMED    19033456
REFERENCE   2  (bases 1 to 6758)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PNAS"; 
            date: "2008-12-9"; volume: "105(49)"; pages: "19264-"
FEATURES             Location/Qualifiers
     source          1..6758
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        827..845
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     827..844
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     858..877
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      933..942
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             939..1655
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2220..2936
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      3007..3016
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     polyA_signal    3020..3154
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3057..3076)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3111..3130
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3223..3580
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(3246..3266)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3431..3566
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3493..3512
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(3600..3617)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3600..3616)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3609..3631)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3829..4284
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3916..3935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4126..4147
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4301..4320)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4455..4477
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4530..4548)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4616..4720
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4721..5581
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRDDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4939..4958)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5752..6340
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6241..6260
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6494..6511
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6664..6694
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6702..6718
                     /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)."
     primer_bind     6707..6729
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6726..6742
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6726..6742
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"