LOCUS       Exported                9204 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPKm-232, complete sequence.
ACCESSION   MG520655
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9204)
  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 9204)
  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 9204)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9204
                     /organism="Cloning vector pPKm-232"
                     /mol_type="other DNA"
                     /db_xref="taxon:2071371"
     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..1350
                     /label=transit peptide mito
                     /note="transit peptide mito"
     CDS             1264..1338
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      1360..1369
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_feature    1369..1398
                     /label=MYC tag codon optimized
                     /note="MYC tag codon optimized"
     CDS             1369..1398
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    1399..2127
                     /label=tHO1
                     /note="tHO1"
     misc_feature    2128..2193
                     /label=P2A peptide
                     /note="P2A peptide"
     CDS             2137..2193
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2194..2268
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     misc_feature    2296..2319
                     /label=FLAG
                     /note="FLAG"
     CDS             2296..2319
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2326..3051
                     /label=tPCYA
                     /note="tPCYA"
     regulatory      2760..2769
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        3089..3418
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     regulatory      3222..3418
                     /regulatory_class="promoter"
                     /note="SV40 early promoter"
     rep_origin      3269..3404
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     misc_feature    3428..4027
                     /label=Puromyocin resistance gene variant
                     /note="Puromyocin resistance gene variant"
     CDS             3428..4027
                     /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    4303..4324
                     /label=U3PPT
                     /note="U3PPT"
     LTR             4320..4553
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    4373..4553
                     /label=HIV-1_3_LTR
                     /note="HIV-1_3_LTR"
     regulatory      4707..4934
                     /regulatory_class="terminator"
                     /note="BGH_PA_terminator"
     polyA_signal    4710..4934
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      4977..4986
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        5030..5134
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5135..5995
                     /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    5333..5992
                     /label=AmpR
                     /note="AmpR"
     rep_origin      6090..6772
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      6166..6754
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     LTR             6913..7546
                     /label=3 LTR
                     /note="3 long terminal repeat (LTR) from HIV-1"
     misc_feature    7366..7546
                     /label=HIV-1_LTR
                     /note="HIV-1_LTR"
     misc_feature    7593..7718
                     /label=HIV-1-psi
                     /note="HIV-1-psi"
     misc_feature    8211..8444
                     /label=RRE
                     /note="RRE"
     misc_feature    8960..9077
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"