LOCUS       40924_34774        7477 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPKm-195, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7477)
  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 7477)
  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 7477)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7477)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol (2018) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (15-NOV-2017) Bioengineering, UCSD, 9500 Gilman drive, PFBH room 251
            MC0412, La Jolla, CA 92093, United States of America"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     promoter        655..673
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    693..2555
                     /label=PhyB
                     /note="PhyB"
     misc_feature    2562..2737
                     /label=MTAD
                     /note="MTAD"
     CDS             2742..2753
                     /codon_start=1
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
     CDS             2773..2793
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     promoter        complement(2821..2839)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     polyA_signal    2865..3089
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3135..3563
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3577..3907
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             3974..4765
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    4942..5075
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5112..5128)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5136..5152)
                     /label=lac operator
                     /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(5160..5190)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5205..5226)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5514..6102)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6276..7133)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7134..7238)
                     /label=AmpR promoter
ORIGIN
        1 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
       61 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      121 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      181 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg actatttacg
      241 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      301 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      361 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      421 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      481 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      541 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      601 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
      661 gactcactat agggagaccc aagcttggta ccatggtttc cggagtcggg ggtagtggcg
      721 gtggccgtgg cggtggccgt ggcggagaag aagaaccgtc gtcaagtcac actcctaata
      781 accgaagagg aggagaacaa gctcaatcgt cgggaacgaa atctctcaga ccaagaagca
      841 acactgaatc aatgagcaaa gcaattcaac agtacaccgt cgacgcaaga ctccacgccg
      901 ttttcgaaca atccggcgaa tcagggaaat cattcgacta ctcacaatca ctcaaaacga
      961 cgacgtacgg ttcctctgta cctgagcaac agatcacagc ttatctctct cgaatccagc
     1021 gaggtggtta cattcagcct ttcggatgta tgatcgccgt cgatgaatcc agtttccgga
     1081 tcatcggtta cagtgaaaac gccagagaaa tgttagggat tatgcctcaa tctgttccta
     1141 ctcttgagaa acctgagatt ctagctatgg gaactgatgt gagatctttg ttcacttctt
     1201 cgagctcgat tctactcgag cgtgctttcg ttgctcgaga gattaccttg ttaaatccgg
     1261 tttggataca ttccaagaat actggtaaac cgttttacgc cattcttcat aggattgatg
     1321 ttggtgttgt tattgattta gagccagcta gaactgaaga tcctgcgctt tctattgctg
     1381 gtgctgttca atcgcagaaa ctcgcggttc gtgcgatttc tcagttacag gctcttcctg
     1441 gtggagatat taagcttttg tgtgacactg tcgtggaaag tgtgagggac ttgactggtt
     1501 atgatcgtgt tatggtttat aagtttcatg aagatgagca tggagaagtt gtagctgaga
     1561 gtaaacgaga cgatttagag ccttatattg gactgcatta tcctgctact gatattcctc
     1621 aagcgtcaag gttcttgttt aagcagaacc gtgtccgaat gatagtagat tgcaatgcca
     1681 cacctgttct tgtggtccag gacgataggc taactcagtc tatgtgcttg gttggttcta
     1741 ctcttagggc tcctcatggt tgtcactctc agtatatggc taacatggga tctattgcgt
     1801 ctttagcaat ggcggttata atcaatggaa atgaagatga tgggagcaat gtagctagtg
     1861 gaagaagctc gatgaggctt tggggtttgg ttgtttgcca tcacacttct tctcgctgca
     1921 taccgtttcc gctaaggtat gcttgtgagt ttttgatgca ggctttcggt ttacagttaa
     1981 acatggaatt gcagttagct ttgcaaatgt cagagaaacg cgttttgaga acgcagacac
     2041 tgttatgtga tatgcttctg cgtgactcgc ctgctggaat tgttacacag agtcccagta
     2101 tcatggactt agtgaaatgt gacggtgcag catttcttta ccacgggaag tattacccgt
     2161 tgggtgttgc tcctagtgaa gttcagataa aagatgttgt ggagtggttg cttgcgaatc
     2221 atgcggattc aaccggatta agcactgata gtttaggcga tgcggggtat cccggtgcag
     2281 ctgcgttagg ggatgctgtg tgcggtatgg cagttgcata tatcacaaaa agagactttc
     2341 ttttttggtt tcgatctcac actgcgaaag aaatcaaatg gggaggcgct aagcatcatc
     2401 cggaggataa agatgatggg caacgaatgc atcctcgttc gtcctttcag gcttttcttg
     2461 aagttgttaa gagccggagt cagccatggg aaactgcgga aatggatgcg attcactcgc
     2521 tccagcttat tctgagagac tcttttaaag aatctggatc catgggatct cctaggtttg
     2581 atactgacct gttcgatacc gacttgtttg atactgacct ttttgatact gacctgttcg
     2641 ataccgactt gtttgatact gacctttttg atactgacct gttcgatacc gacttgtttg
     2701 atactgacct ttttgatact gacctgttcg ataccgactt gatcgaggga aggggtggcg
     2761 aattaattca tgcctaagaa aaagagaaag gtgggcggag gcgctagtag agggccctat
     2821 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     2881 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2941 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3001 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3061 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3121 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3181 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3241 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg gcatcccttt
     3301 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3361 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3421 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3481 ttcttttgat ttataaggga ttttggggat ttcggcctat tggttaaaaa atgagctgat
     3541 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3601 tccccaggct ccccaggcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3661 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3721 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3781 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3841 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3901 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     3961 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4021 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4081 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4141 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4201 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4261 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4321 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4381 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4441 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4501 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4561 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4621 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4681 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4741 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4801 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4861 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4921 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4981 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5041 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5101 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5161 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5221 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5281 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5341 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5401 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5461 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5521 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5581 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5641 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5701 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5761 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5821 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5881 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5941 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6001 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6061 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6121 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6181 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6241 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6301 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6361 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6421 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6481 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6541 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6601 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6661 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6721 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6781 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6841 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6901 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6961 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7021 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7081 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7141 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7201 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7261 cctgacgtcg acggatcggg agatctcccg atcccctatg gtcgactctc agtacaatct
     7321 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     7381 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     7441 agaatctgct tagggttagg cgttttgcgc tgcttcg
//
