LOCUS       Exported                9018 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPKm-243, complete sequence.
ACCESSION   MG520662
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9018)
  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 9018)
  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 9018)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9018
                     /organism="Cloning vector pPKm-243"
                     /mol_type="other DNA"
                     /db_xref="taxon:2071378"
     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"
     regulatory      1258..1267
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_feature    1264..1971
                     /label=mOrange
                     /note="mOrange"
     CDS             1264..1968
                     /codon_start=1
                     /product="orange monomeric derivative of DsRed fluorescent 
                     protein (Shanet et al., 2004)"
                     /label=mOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKMRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVGIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELY"
     misc_feature    1972..2037
                     /label=P2A peptide
                     /note="P2A peptide"
     CDS             1981..2037
                     /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"
     misc_feature    2038..2124
                     /label=transit peptide MTS
                     /note="transit peptide MTS"
     CDS             2038..2112
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     misc_feature    2140..2157
                     /label=HIS tag
                     /note="HIS tag"
     CDS             2140..2157
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    2170..2883
                     /label=superfolder GFP
                     /note="superfolder GFP"
     CDS             2170..2883
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     promoter        2903..3232
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     regulatory      3036..3232
                     /regulatory_class="promoter"
                     /note="SV40 early promoter"
     rep_origin      3083..3218
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     misc_feature    3242..3841
                     /label=Puromyocin resistance gene variant
                     /note="Puromyocin resistance gene variant"
     CDS             3242..3841
                     /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    4117..4138
                     /label=U3PPT
                     /note="U3PPT"
     LTR             4134..4367
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    4134..4186
                     /label=delta_U3
                     /note="delta_U3"
     misc_feature    4187..4367
                     /label=truncated 5/3LTR
                     /note="truncated 5/3LTR"
     misc_feature    4187..4367
                     /label=HIV-1_LTR
                     /note="HIV-1_LTR"
     misc_feature    4187..4367
                     /label=HIV-1_3_LTR(1)
                     /note="HIV-1_3_LTR(1)"
     regulatory      4521..4748
                     /regulatory_class="terminator"
                     /note="BGH_PA_terminator"
     polyA_signal    4524..4748
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      4791..4800
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        4844..4948
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      4879..4907
                     /regulatory_class="promoter"
                     /note="AMP promoter"
     CDS             4949..5809
                     /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    5147..5806
                     /label=AmpR
                     /note="AmpR"
     rep_origin      5904..6586
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      5980..6568
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     LTR             6727..7360
                     /label=3 LTR
                     /note="3 long terminal repeat (LTR) from HIV-1"
     misc_feature    7180..7360
                     /label=truncated 5/3LTR(1)
                     /note="truncated 5/3LTR(1)"
     misc_feature    7180..7360
                     /label=HIV-1_LTR(1)
                     /note="HIV-1_LTR(1)"
     misc_feature    7180..7360
                     /label=HIV-1_3_LTR
                     /note="HIV-1_3_LTR"
     misc_feature    7407..7532
                     /label=HIV-1-psi
                     /note="HIV-1-psi"
     misc_feature    8025..8258
                     /label=RRE
                     /note="RRE"
     misc_feature    8774..8891
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"