LOCUS       40924_42714        8857 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle vector ptCC9, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8857)
  AUTHORS   Stukenberg D, Zauner S, Dell'Aquila G, Maier UG.
  TITLE     Optimizing CRISPR/Cas9 for the Diatom Phaeodactylum tricornutum
  JOURNAL   Front Plant Sci 9, 740 (2018)
  PUBMED    29928285
REFERENCE   2  (bases 1 to 8857)
  AUTHORS   Stuckenberg D, Zauner S.
  TITLE     Direct Submission
  JOURNAL   Submitted (29-MAR-2018) Philipps-University Marburg, Cellbiology, 
            Karl-von-Frisch Str. 8, Marburg 35043, Deutschland
REFERENCE   3  (bases 1 to 8857)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8857)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Front Plant
            Sci 9, 740 (2018)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (29-MAR-2018) Philipps-University Marburg, Cellbiology, 
            Karl-von-Frisch Str. 8, Marburg 35043, Deutschland"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1..415
                     /note="nitrate reductase; derived from Phaeodactylum 
                     tricornutum"
                     /regulatory_class="promoter"
     CDS             431..496
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             503..523
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             548..4648
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             4649..4696
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     regulatory      4700..4972
                     /note="nitrate reductase; derived from Phaeodactylum 
                     tricornutum"
                     /regulatory_class="terminator"
     regulatory      5010..5288
                     /note="U6-RNA promoter; derived from Phaeodactylum
                     tricornutum"
                     /regulatory_class="promoter"
     misc_RNA        5310..5385
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      5386..5723
                     /note="U6-RNA terminator; derived from Phaeodactylum 
                     tricornutum"
                     /regulatory_class="terminator"
     regulatory      5724..5916
                     /note="fcpB promoter; derived from Phaeodactylum
                     tricornutum"
                     /regulatory_class="promoter"
     CDS             5917..6288
                     /label=BleoR
                     /note="antibiotic-binding protein"
     regulatory      6292..6532
                     /note="fcpB teminator; derived from Phaeodactylum
                     tricornutum"
                     /regulatory_class="terminator"
     promoter        complement(6543..6561)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(6819..7407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7581..8438)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8439..8543)
                     /label=AmpR promoter
ORIGIN
        1 ggaagtgact gtaaacgaga agtgcacgaa gccttttctt gtgacgtcac aaaccgaaca
       61 gccctacgtg gcgtgcgact ttccggcact ttggatatcg ttgccctata tgtattgggt
      121 gtatacatgt cgtattccaa ctacgacaga aaacagtcag ttacgggtat tcgaacttcc
      181 ggcacctgca gcgaggagtt ttgtgtccgt cgcacatcct ccgtgctttc ggcacctacg
      241 taggcggcaa acttcctgcc ttccgcccgc cttgcggcat tccggctccg ggccagaatt
      301 gcccgggtgt tcacaatttt gcctcctcac gaaaaaacgt tctactttgt attttggtcg
      361 ggtttcggat ccttccagca accattctca ttcaaagtca ccacttgtgc gaacgatggc
      421 ctctagaatg gactataagg accacgacgg agattacaag gatcatgata ttgattacaa
      481 agacgatgat gataaaatgg ccccaaagaa gaagcggaaa gtcggtatac acggagtccc
      541 agcagccgac aagaagtact cgattggatt ggacatcggc accaactctg ttggatgggc
      601 cgtcatcacg gacgagtaca aggtgcccag caagaaattc aaagtactgg gaaacactga
      661 ccgccattcg atcaagaaga atttgattgg agctttgctg tttgacagcg gcgaaacagc
      721 ggaagccacg cggcttaaac gaaccgcgag acgaagatat acgcgacgga agaaccgtat
      781 ctgctatcta caagagattt tctcgaatga gatggcaaaa gtcgatgact ctttctttca
      841 ccgattggaa gaatccttcc tcgttgaaga ggataagaaa catgagcggc acccgatatt
      901 tggaaacatt gtggacgagg tggcttacca tgagaaatac ccgactattt accacctgcg
      961 aaagaaattg gtggactcga cggacaaagc ggatcttcgc ctaatctatt tggcgctagc
     1021 acatatgatt aagtttcgcg gccacttctt gatcgaaggc gacttgaatc cggacaacag
     1081 tgatgttgac aaactgttta ttcaacttgt acaaacttac aaccagctct ttgaagaaaa
     1141 tcccattaac gcttcgggag tggacgcgaa agccatcctt tctgcccgac tcagcaagag
     1201 ccgacgccta gaaaatttga ttgcgcaact tcccggcgag aaaaagaatg gactttttgg
     1261 aaatcttatt gctttgtcgc tgggccttac gccgaacttt aagagcaact tcgaccttgc
     1321 ggaagatgcc aaattgcaac tgagtaaaga cacatatgac gacgacctcg acaatctttt
     1381 ggcacaaata ggagatcagt acgccgacct ctttttggcg gctaagaact tgtccgacgc
     1441 cattctgttg tcggacatcc ttcgagttaa tacggagatt actaaggcgc ccttgagcgc
     1501 ctctatgatc aaaagatacg acgagcatca ccaagacctg accctcctta aagctctcgt
     1561 ccggcaacag ctgcctgaga agtacaaaga gattttcttt gatcaaagca aaaacggata
     1621 cgcgggctac attgacgggg gagcctcgca agaagagttc tacaagttta ttaaaccgat
     1681 cctggaaaag atggacggca cggaggaatt actcgtgaaa cttaaccgag aggacctttt
     1741 gcgtaagcaa cggactttcg acaacggatc tattccgcac caaatccatc tcggagaatt
     1801 gcacgcaatt ctgcgccggc aagaagattt ttatccattt ctcaaggaca accgggaaaa
     1861 gattgagaaa atcttaacct tccgcatacc ctactacgtc ggccctttgg ccaggggaaa
     1921 ttcgagattt gcctggatga cgcgaaagag tgaggaaact attactccct ggaatttcga
     1981 agaagtggta gacaaaggag cttccgcgca atcctttatc gagcgcatga ccaacttcga
     2041 taagaacctc ccgaatgaga aagtcctacc caagcattcg ttgctctacg aatactttac
     2101 ggtgtataat gagctgacta aagtcaaata cgttacagag ggaatgcgaa aaccggcttt
     2161 cctctcggga gagcagaaaa aggccatagt cgatcttctc tttaaaacga accgtaaagt
     2221 taccgtgaaa caactaaaag aggactactt caagaaaatc gaatgctttg actccgttga
     2281 aatttccggc gttgaagatc ggttcaatgc ctccttggga acataccacg atctcctgaa
     2341 aattatcaaa gacaaggact ttcttgacaa tgaggaaaac gaggacattc ttgaagatat
     2401 tgtcttgacg ctaacacttt ttgaggatcg agagatgatt gaggaacgcc ttaaaaccta
     2461 tgcgcatctg ttcgacgaca aagttatgaa gcaattgaag cgccggagat atacgggatg
     2521 gggaaggttg agtcggaaac tgataaatgg catacgcgat aagcagtccg gcaaaacaat
     2581 tctcgatttc ttgaagtccg acggatttgc gaatcgaaac ttcatgcaac tgatccatga
     2641 cgattcgttg acctttaaag aggacattca aaaagcccaa gtatccggac aaggagattc
     2701 tttgcacgag catattgcga atcttgctgg aagtccggct attaaaaagg gcattctcca
     2761 gacagtaaag gtcgtggatg aactcgttaa agttatgggc cgacacaaac ccgagaacat
     2821 agtcatcgaa atggcgagag agaaccagac cacccaaaag ggacagaaaa attcacgcga
     2881 gcgaatgaag cggattgaag aggggatcaa agagcttggc agccaaattc tgaaagaaca
     2941 cccggttgaa aacacccaat tgcagaacga gaagctctac ctgtactatc ttcaaaatgg
     3001 gcgtgatatg tacgtcgatc aagaattgga catcaaccgg ctatccgatt acgatgtgga
     3061 ccatattgtt cctcagtcgt ttctcaaaga cgactccata gacaataagg ttctaacgag
     3121 aagcgacaag aaccgcggca aatcagacaa cgtcccctcc gaggaagtag ttaagaaaat
     3181 gaagaattac tggcgccaat tgctgaacgc taagctcatt acccaaagaa agtttgacaa
     3241 tttgacgaaa gccgagcgag gcgggctctc ggaactggat aaggccggct tcattaaaag
     3301 acaattggtg gaaacgcgac aaatcacaaa gcacgtcgca cagattctcg actcccgcat
     3361 gaacactaag tatgacgaga atgacaaact aatccgggaa gtcaaagtga ttaccttgaa
     3421 gtccaagctg gtttccgatt ttcgtaaaga ttttcagttt tacaaagttc gcgagatcaa
     3481 caactaccat cacgcgcacg acgcttactt gaacgctgtc gtgggaacgg ccttgattaa
     3541 aaaataccct aagctggaaa gcgagttcgt ttacggcgat tacaaggtgt atgacgtacg
     3601 caaaatgatc gcgaagtcag agcaggaaat tggcaaagct actgcgaagt acttctttta
     3661 tagcaacata atgaattttt tcaaaacgga gattaccctc gctaacggcg agattcgcaa
     3721 gcgccctctt atcgagacaa acggcgaaac gggggagatt gtctgggata aaggccgcga
     3781 ttttgcgact gtgcggaaag tcttgtcgat gccgcaagtg aatatagtca aaaagacgga
     3841 ggtgcaaaca ggcggctttt cgaaagaatc tatccttccc aaaaggaact ctgataagtt
     3901 gattgcgcga aaaaaggatt gggaccctaa gaagtatggc ggctttgaca gcccgaccgt
     3961 agcatattct gtccttgtgg tggccaaagt cgaaaagggc aaatccaaga aactgaagag
     4021 tgtgaaagaa ttgcttggga ttacgatcat ggaaagatca tcttttgaga agaatcccat
     4081 cgactttctc gaagctaaag gctacaaaga agtgaaaaag gacctgatta tcaagttgcc
     4141 taagtactcc ttgtttgagc tggaaaatgg ccgtaagcga atgctcgctt ctgccggcga
     4201 actccagaag ggaaacgaat tggcgctgcc ctccaaatat gttaactttc tttaccttgc
     4261 ctcgcactat gagaagctca agggctcccc cgaggataat gagcaaaaac aactgtttgt
     4321 agaacagcat aagcactact tggacgagat cattgagcaa attagcgagt tctccaagcg
     4381 agtcatcctt gccgacgcta atctggacaa agtcctgtcc gcctacaata agcaccgcga
     4441 taagccaatc cgagagcaag cggagaatat tatccacttg tttacgctca ccaatcttgg
     4501 agcccctgcc gcctttaagt actttgacac caccatcgac cgcaagaggt acaccagcac
     4561 caaagaagtc ttggacgcca ccctcattca ccaatcaatc accggcttgt acgagacacg
     4621 catcgacctg tctcaactcg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca
     4681 ggcaaaaaag aaaaagtaac aatttacttt aagactagcg caattttttt cttacattag
     4741 atgaacataa aacgacgatg aggatcactg ttagggaaac gatgaactac gctgaggact
     4801 gcctattatt cgtcctttga tggtgtggtt gttctttggt ttgcggccga gcgcaacgac
     4861 caaggcctgt accaaaaacg aagttgataa tcagatattt gagttgtaac aagaaactaa
     4921 agcattattt tagcagtagc acgaatcagg atctaaaatg aacgcaattg catggttgat
     4981 gtctaagctg cgttagaact tagcatatgg ttggctcgga agttggtgtt gacggtgagc
     5041 tggaaattgg ttgtcggtca ctgctagcga gaagaaaacg gaggacagaa ggaagtgaaa
     5101 ctcggttcgt tctcgacagc ctcactgtca atatgctcat tttcaatcct tagcgctttt
     5161 aatgtcgaat tgacggtaaa ttgaatagga tctataatat ctacaaggta ctttgacacg
     5221 ccaagtattc attgttagtc aacaatattt tagagcttta taaggtcaaa aaacaccttc
     5281 aaagtcgagg agaccgagag agggtctcag ttttagagct agaaatagca agttaaaata
     5341 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tttagaaccg
     5401 ctcacccatg ctatcgtatg tcatttacat tgacaaactg ctatgaatgc gtttgcgata
     5461 acttcagctc gctgaagccc tagacatttc gtttgactaa attgttttcc ggttgcttta
     5521 ctaccgaccg gtctgcgggt agcatgtctt ggcttttgcg agttcgagag aatttatgga
     5581 caagtgcgtt tacagtttgg aacacgagtg tgagtgagcg gaattggcta tctataacat
     5641 tcttatttca ggaggctggt gattagcaga attttcaaag atctcgaatc taaaatcgcc
     5701 atgggctcga cgaacttttc tgctccggag agattcaaaa ttcaatctgt ttggacctgg
     5761 ataagacaca agagcgacat cctgacatga acgccgtaaa cagcaaatcc tggttgaaca
     5821 cgtatccttt tgggggcctc ccgctacgac gctcgctcca gctggggctt ccttactata
     5881 cacagcgcgc atatttcacg gttgccagat gtcaagatgg ccaagttgac cagtgccgtt
     5941 ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg
     6001 ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg
     6061 ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg
     6121 cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac
     6181 gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg
     6241 cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg aaccttcctt
     6301 aaaaatttaa ttttcattag ttgcagtcac tccgctttgg tttcacagtc aggaataaca
     6361 ctagctcgtc ttcaccatgg atgccaatct cgcctattca tggtgtataa aagttcaaca
     6421 tccaaagcta gaacttttgg aaagagaaag aatatccgaa tagggcacgg cgtgccgtat
     6481 tgttggagtg gactagcaga aagtgaggaa ggcacaggat gagttttctc gaggccagtc
     6541 tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc
     6601 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     6661 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6721 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     7021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     7081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     7141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     7201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     7261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     7321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     7381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     7441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     7501 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     7561 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     7621 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     7681 ggcttaccat ctggccccag tgctgcaatg ataccgcgag atccacgctc accggctcca
     7741 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7801 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7861 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7921 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7981 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     8041 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     8101 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     8161 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     8221 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     8281 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     8341 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     8401 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     8461 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8521 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     8581 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     8641 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     8701 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     8761 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     8821 catatgggca ctagaaagga aggaagcaaa gacgaac
//
