LOCUS       Exported               14389 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  expresses mouse Piezo1 with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    mPiezo1-IRES-eGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14389)
  AUTHORS   Coste B, Mathur J, Schmidt M, Earley TJ, Ranade S, Petrus MJ, Dubin 
            AE, Patapoutian A
  TITLE     Piezo1 and Piezo2 are essential components of distinct mechanically 
            activated cation channels.
  JOURNAL   Science. 2010 Oct 1;330(6000):55-60. doi: 10.1126/science.1193270. 
            Epub 2010 Sep 2.
  PUBMED    20813920
REFERENCE   2  (bases 1 to 14389)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1126/science.1193270"; journalName: "Science"; date: "2010-10-1-
            1"; volume: "330"; issue: "6000"; pages: "55-60"
FEATURES             Location/Qualifiers
     source          1..14389
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        124..503
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        504..707
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     658..678
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     752..771
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        752..770
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      823..832
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_feature    8528..9114
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(8695..8712)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     8922..8941
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             9115..9834
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             9115..9831
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(9160..9181)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(9421..9440)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9768..9789
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(9869..9886)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9875..10099
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10145..10573
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10232..10251)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10442..10463
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10582..10602)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10587..10916
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10767..10902
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10829..10848
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             10983..11777
                     /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(11037..11056)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     11647..11666
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    11951..12072
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11988..12007)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12042..12061
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12121..12137)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12121..12137)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12134..12156)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12145..12161
                     /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(12169..12199)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12214..12235
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12352..12369)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12523..13111)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12603..12622)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(13282..14142)
                     /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     13905..13924
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14143..14247)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(14322..14341)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"