LOCUS       40924_34779        6195 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPKm-196, complete sequence.
ACCESSION   .
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)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6195)
  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..6195
                     /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..992
                     /label=PIF6
                     /note="PIF6"
     CDS             1005..1025
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             1035..1475
                     /codon_start=1
                     /label=GAL4 DNA binding domain
                     /note="DNA binding domain of the GAL4 transcriptional
                     activator"
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             1491..1517
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     promoter        complement(1539..1557)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     polyA_signal    1583..1807
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1853..2281
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2295..2625
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2692..3483
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3660..3793
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3830..3846)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3854..3870)
                     /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(3878..3908)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3923..3944)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4232..4820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4994..5851)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5852..5956)
                     /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 ccatgatgtt cttaccaacc gattattgtt
      721 gcaggttaag cgatcaagag tatatggagc ttgtgtttga gaatggccag attcttgcaa
      781 agggccaaag atccaacgtt tctctgcata atcaacgtac caaatcgatc atggatttgt
      841 atgaggcaga gtataacgag gatttcatga agagtatcat ccatggtggt ggtggtgcca
      901 tcacaaatct cggggacacg caggttgttc cacaaagtca tgttgctgct gcccatgaaa
      961 caaacatgtt ggaaagcaat aaacatgttg acacgcggcc gcaacctaag aaaaagagaa
     1021 aggtgggcgg aggcatgaag ctactgtctt ctatcgaaca agcatgcgat atttgccgac
     1081 ttaaaaagct caagtgctcc aaagaaaaac cgaagtgcgc caagtgtctg aagaacaact
     1141 gggagtgtcg ctactctccc aaaaccaaaa ggtctccgct gactagggca catctgacag
     1201 aagtggaatc aaggctagaa agactggaac agctatttct actgattttt cctcgagaag
     1261 accttgacat gattttgaaa atggattctt tacaggatat aaaagcattg ttaacaggat
     1321 tatttgtaca agataatgtg aataaagatg ccgtcacaga tagattggct tcagtggaga
     1381 ctgatatgcc tctaacattg agacagcata gaataagtgc gacatcatca tcggaagaga
     1441 gtagtaacaa aggtcaaaga cagttgactg tatcgccgga attcatggct tacccatacg
     1501 atgttccaga ttacgcttga taatctagag ggccctattc tatagtgtca cctaaatgct
     1561 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     1621 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     1681 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     1741 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     1801 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     1861 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1921 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1981 ggctttcccc gtcaagctct aaatcggggc atccctttag ggttccgatt tagtgcttta
     2041 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2101 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2161 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2221 ttggggattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2281 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccaggcaggc
     2341 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2401 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     2461 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2521 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     2581 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     2641 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     2701 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     2761 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     2821 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     2881 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     2941 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3001 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3061 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3121 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3181 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3241 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3301 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3361 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3421 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3481 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     3541 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     3601 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     3661 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     3721 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     3781 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     3841 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3901 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3961 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4021 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4081 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4141 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4201 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4261 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4321 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4381 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
     4441 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4501 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4561 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4621 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     4681 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4741 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4801 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4861 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4921 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4981 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5041 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5101 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5161 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5221 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5281 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5341 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5401 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5461 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5521 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5581 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5641 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5701 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5761 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5821 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5881 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5941 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     6001 atctcccgat cccctatggt cgactctcag tacaatctgc tctgatgccg catagttaag
     6061 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     6121 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     6181 ttttgcgctg cttcg
//
