LOCUS       40924_34869        6262 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPKm-293, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6262)
  AUTHORS   Kyriakakis P, Catanho M, Hoffner N, Thavarajah W, Hu VJ, Chao SS, 
            Hsu A, Pham V, Naghavian L, Dozier LE, Patrick GN, Coleman TP.
  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 6262)
  AUTHORS   Kyriakakis P.
  TITLE     Direct Submission
  JOURNAL   Submitted (30-NOV-2017) Bioengineering, UCSD, 9500 Gilman Drive, 
            PFBH Room 251 MC0412, La Jolla, CA 92093, USA
REFERENCE   3  (bases 1 to 6262)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6262)
  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 
            (30-NOV-2017) Bioengineering, UCSD, 9500 Gilman Drive, PFBH Room 251
            MC0412, La Jolla, CA 92093, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6262
                     /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"
     CDS             696..716
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             720..1340
                     /codon_start=1
                     /label=TetR
                     /note="tetracycline repressor TetR"
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     misc_feature    1347..1522
                     /label=MTAD
                     /note="MTAD"
     CDS             1527..1538
                     /codon_start=1
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
     CDS             1558..1578
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     promoter        complement(1606..1624)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     polyA_signal    1650..1874
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1920..2348
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2362..2692
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2759..3550
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3727..3860
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3897..3913)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3921..3937)
                     /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(3945..3975)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3990..4011)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4299..4887)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5061..5918)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5919..6023)
                     /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 ccatgcctaa gaaaaagaga aaggtgggca
      721 tgtcaagatt agataaaagt aaagtgatta acagcgcatt agagctgctt aatgaggtcg
      781 gaatcgaagg tttaacaacc cgtaaactcg cccagaagct aggtgtagag cagcctacat
      841 tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt gagatgttag
      901 ataggcacca tactcacttt tgccctttag aaggggaaag ctggcaagat tttttacgta
      961 ataacgctaa aagttttaga tgtgctttac taagtcatcg ggatggagca aaagtacatt
     1021 taggtacacg gcctacagaa aaacagtatg aaactctcga aaatcaatta gcctttttat
     1081 gccaacaagg tttttcacta gagaatgcat tatatgcact cagcgctgtg gggcatttta
     1141 ctttaggttg cgtattggaa gatcaagagc atcaagtcgc taaagaagaa agggaaacac
     1201 ctactactga tagtatgccg ccattattac gacaagctat cgaattattt gatcaccaag
     1261 gtgcagagcc agccttctta ttcggccttg aattgatcat ctgcggatta gaaaaacaac
     1321 ttaaatgtga aagtgggtcc ggatccatgg gatctcctag gtttgatact gacctgttcg
     1381 ataccgactt gtttgatact gacctttttg atactgacct gttcgatacc gacttgtttg
     1441 atactgacct ttttgatact gacctgttcg ataccgactt gtttgatact gacctttttg
     1501 atactgacct gttcgatacc gacttgatcg agggaagggg tggcgaatta attcatgcct
     1561 aagaaaaaga gaaaggtggg cggaggcgct agtagagggc cctattctat agtgtcacct
     1621 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     1681 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1741 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1801 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     1861 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     1921 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1981 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2041 gttcgccggc tttccccgtc aagctctaaa tcggggcatc cctttagggt tccgatttag
     2101 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2161 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2221 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2281 agggattttg gggatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2341 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2401 ggcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2461 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2521 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2581 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     2641 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     2701 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     2761 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     2821 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     2881 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     2941 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3001 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3061 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3121 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3181 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3241 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     3301 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3361 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3421 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3481 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3541 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     3601 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3661 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3721 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3781 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3841 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     3901 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3961 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4021 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4081 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4141 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4201 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4261 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4321 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4381 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4441 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     4501 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4561 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4621 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4681 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     4741 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4801 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4861 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4921 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4981 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5041 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5101 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5161 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5221 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5281 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5341 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5401 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5461 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5521 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5581 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5641 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5701 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5761 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5821 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5881 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5941 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6001 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     6061 tcgggagatc tcccgatccc ctatggtcga ctctcagtac aatctgctct gatgccgcat
     6121 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     6181 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     6241 ttaggcgttt tgcgctgctt cg
//
