LOCUS       Exported               12253 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPKm-230, complete sequence.
ACCESSION   MG520653
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12253)
  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 12253)
  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 12253)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..12253
                     /organism="Cloning vector pPKm-230"
                     /mol_type="other DNA"
                     /db_xref="taxon:2070799"
     regulatory      1..1179
                     /regulatory_class="promoter"
                     /note="ef1-alpha promoter"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     misc_feature    1264..2833
                     /label=PIF3-1-523
                     /note="PIF3-1-523"
     misc_feature    2842..3017
                     /label=MTAD
                     /note="MTAD"
     CDS             3022..3033
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    3049..3072
                     /label=SV40NLS
                     /note="SV40NLS"
     CDS             3049..3069
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3082..3108
                     /label=HA tag
                     /note="HA tag"
     CDS             3082..3108
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3121..3707
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    join(3121..3599,3601..3703)
                     /label=IRES
                     /note="IRES"
     misc_feature    3720..5584
                     /label=PhyB
                     /note="PhyB"
     misc_feature    5595..5618
                     /label=SV40NLS(1)
                     /note="SV40NLS(1)"
     CDS             5595..5615
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    5625..6070
                     /label=GBD
                     /note="GBD"
     CDS             5625..6065
                     /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"
     promoter        6138..6467
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     regulatory      6271..6410
                     /regulatory_class="promoter"
                     /note="SV40 early promoter"
     rep_origin      6318..6453
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      6411..6467
                     /regulatory_class="promoter"
                     /note="SV40 early promoter(1)"
     misc_feature    6477..7076
                     /label=Puromyocin resistance gene variant
                     /note="Puromyocin resistance gene variant"
     CDS             6477..7076
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     misc_feature    7352..7373
                     /label=U3PPT
                     /note="U3PPT"
     LTR             7369..7602
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    7422..7602
                     /label=truncated 5/3LTR
                     /note="truncated 5/3LTR"
     misc_feature    7422..7602
                     /label=HIV-1_LTR
                     /note="HIV-1_LTR"
     regulatory      7756..7983
                     /regulatory_class="terminator"
                     /note="BGH_PA_terminator"
     polyA_signal    7759..7983
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      8026..8035
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        8079..8183
                     /gene="bla"
                     /label=AmpR promoter
     CDS             8184..9044
                     /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    8382..9041
                     /label=AmpR
                     /note="AmpR"
     rep_origin      9139..9821
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      9215..9803
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     LTR             9962..10595
                     /label=3 LTR
                     /note="3 long terminal repeat (LTR) from HIV-1"
     misc_feature    10415..10595
                     /label=truncated 5/3LTR(1)
                     /note="truncated 5/3LTR(1)"
     misc_feature    10415..10595
                     /label=HIV-1_LTR(1)
                     /note="HIV-1_LTR(1)"
     misc_feature    10642..10767
                     /label=HIV-1-psi
                     /note="HIV-1-psi"
     misc_feature    11260..11493
                     /label=RRE
                     /note="RRE"
     misc_feature    12009..12126
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"