LOCUS       Exported                6195 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPKm-196, complete sequence.
ACCESSION   MG520649
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6195)
  AUTHORS   Kyriakakis P, Catanho M, Hoffner N, Thavarajah W, Jian-Yu V, Chao 
            SS, Hsu A, Pham V, Naghavian L, Dozier LE, Patrick G, Coleman T.
  TITLE     Biosynthesis of Orthogonal Molecules Using Ferredoxin and 
            Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB 
            Optogenetics
  JOURNAL   ACS Synth Biol (2018) In press
  PUBMED    29301067
REFERENCE   2  (bases 1 to 6195)
  AUTHORS   Kyriakakis P.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-NOV-2017) Bioengineering, UCSD, 9500 Gilman drive, 
            PFBH room 251 MC0412, La Jolla, CA 92093, United States of America
REFERENCE   3  (bases 1 to 6195)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6195
                     /organism="Cloning vector pPKm-196"
                     /mol_type="other DNA"
                     /db_xref="taxon:2071366"
     regulatory      1..655
                     /regulatory_class="promoter"
                     /note="P cmv"
     enhancer        27..406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        407..610
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     655..674
                     /label=T7
                     /note="T7"
     promoter        655..673
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    693..992
                     /label=PIF6
                     /note="PIF6"
     misc_feature    1005..1028
                     /label=SV40NLS
                     /note="SV40NLS"
     CDS             1005..1025
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    1035..1480
                     /label=GBD
                     /note="GBD"
     CDS             1035..1475
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     misc_feature    1491..1517
                     /label=HA tag
                     /note="HA tag"
     CDS             1491..1517
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1539..1557)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1540..1557)
                     /label=SP6
                     /note="SP6"
     polyA_signal    1583..1807
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    1659..1790
                     /label=BGH pA
                     /note="BGH pA"
     rep_origin      1853..2281
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     rep_origin      1870..2176
                     /label=F1 ori
                     /note="F1 ori"
     promoter        2295..2625
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2476..2611
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2692..3486
                     /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"
     misc_feature    2695..3486
                     /label=Kan/neoR
                     /note="Kan/neoR"
     polyA_signal    3660..3781
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3826..3846)
                     /label=M13-rev
                     /note="M13-rev"
     primer_bind     complement(3830..3846)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_binding    complement(3852..3874)
                     /label=LacO binding site
                     /bound_moiety="LacO"
     protein_bind    3854..3870
                     /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(3878..3908)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3923..3944
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4214..4896)
                     /direction=LEFT
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      complement(4232..4820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4991..5851)
                     /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"
     misc_feature    complement(4994..5653)
                     /label=AmpR
                     /note="AmpR"
     promoter        complement(5852..5956)
                     /gene="bla"
                     /label=AmpR promoter