LOCUS       40924_36983        9953 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRH003, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9953)
  AUTHORS   Wang Y, Wei D, Zhu X, Pan J, Zhang P, Huo L, Zhu X.
  TITLE     A 'suicide' CRISPR-Cas9 system to promote gene deletion and 
            restoration by electroporation in Cryptococcus neoformans
  JOURNAL   Sci Rep 6, 31145 (2016)
  PUBMED    27503169
REFERENCE   2  (bases 1 to 9953)
  AUTHORS   Wang Y, Wei D, Zhu X.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-OCT-2016) Microbiology, Nankai University, No. 94 
            Weijin Road, Tianjin, Tianjin 300071, P.R. China
REFERENCE   3  (bases 1 to 9953)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9953)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep 6, 
            31145 (2016)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (10-OCT-2016) Microbiology, Nankai University, No. 94 Weijin Road, 
            Tianjin, Tianjin 300071, P.R. China"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        407..511
                     /label=AmpR promoter
     CDS             512..1369
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1543..2131
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        2172..2190
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_RNA        complement(2235..2310)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    complement(2311..2328)
                     /label=double BspQI sites
                     /note="double BspQI sites"
     regulatory      complement(2329..2602)
                     /label=CnU6 promoter
                     /note="CnU6 promoter"
                     /regulatory_class="promoter"
     gene            2633..4571
                     /gene="URA5"
                     /label=URA5
     primer_bind     complement(4573..4589)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      4584..5390
                     /label=actin promoter
                     /note="actin promoter"
                     /regulatory_class="promoter"
     regulatory      5385..5394
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5394..5459
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5466..5486
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5511..9611
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     misc_feature    9612..9659
                     /label=Region: NLS
                     /note="Region: NLS"
     CDS             9612..9659
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    9693..9900
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(9934..9952)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
       61 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
      121 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
      181 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
      241 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
      301 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
      361 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
      421 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
      481 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
      541 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
      601 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
      661 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
      721 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
      781 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
      841 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
      901 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
      961 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     1021 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     1081 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1141 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1201 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga
     1261 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     1321 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     1381 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     1441 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     1501 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1561 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1621 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     1681 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     1741 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     1801 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     1861 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     1921 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     1981 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2041 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2101 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2161 cctggccttt taattaaccc tcactaaagg gaacaaaagc tggtaccggg ccccccctcg
     2221 agtaaaacaa aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt
     2281 attttaactt gctatttcta gctctaaaac tgaagagcta gctcttcgca acagtatacc
     2341 ctgccggtgt atgtgcaatt tccactttgg cggataacgg aagcaaataa gacggcggtc
     2401 cacgccgtct cagtgttttt ttattttttt cccgtcatcc attgtttttc tcctcacctc
     2461 ttcattctca acaaatggtt ataattatcg gtcacacctg caggttacca ttgtccttgc
     2521 acgtatactt tgcatgcatg ccgatcaatt taaaaaataa atgaactgta ataatcatgc
     2581 tttgttttta gttctaatgc aaatcgataa gcttgatatc gaattcctgc aggaccaacc
     2641 ctggcacatc aagcggcttc gacatcatct tgtgtccaga tccggccatt ggcttggaag
     2701 atagttcaga gtttgattgg acgatgattg tcttaagagg tggaggcaat ttggggcgac
     2761 gacccattag gtccaggtgc tccagcttgc ctccaggagg tgggagggat gggaccaggg
     2821 agtcatcgag gaagacggaa atggggggag caggagatat gaagtatgat tgggattgag
     2881 aggaagagaa ggggagtttc ctgctaacca ttctgggtca acactaataa aattgtatta
     2941 ttgggtcatt gaagtcagta ttgacaaagt agatacgatg aagattgaca gccaacaatc
     3001 atattacagc ttcctttgtc gcctcaggac gcctgcctgt acttaagttc ctttgataca
     3061 agcttacaaa cttgtaatat gtcggaactt gattagctag acctaaaccc gtgcatttaa
     3121 gaagaaaacc cttaagacct ctgaacaccg tactccgccc tgtacttcct gacctcttgc
     3181 agctcctttt cccacttgcc ggagctttct aagtacttca caatgtcgtc caaaccaata
     3241 ataggctcga caggcacacc gaactcttcc tcaacctcct gtacggtact cttgccgcta
     3301 ccgctctggc ctttctcttg tctgtcgaca agctggacaa ttccgacaag cttcgcttca
     3361 ggggaggcct tgagaatgtc aatagcttca cggatggcct tgccagaggt gagaacatcg
     3421 tcgatgatga cgattcgtcc cttgagaggc gcaccgacca tagtaccgcc ctcaccgtgc
     3481 tactggctta tgagctcatc gctgtcgcac tggttaggac agactcacgt ccttcttctc
     3541 cttcctgttg tagcagtagc cgatatcttt gccggtttgc tgataaaggc ttacagcgga
     3601 gacagcagcc aaggagatac ccttgtaagc tgggccgaag aggacgtcaa agtcaggaat
     3661 cctagaagag gaaagtacct tggcgtaagc ctgagcggta gttgagagaa gcgatgaaga
     3721 gtaaaggaga ccggcattga agaagtaagg ggattggctg aacacatgtc agattcgatt
     3781 ggactgtgaa gcgctgcaat atggctcacc ggccggactt caaggtaaag ttgccgaaaa
     3841 gaagcacgcc atgttcgatg gcagcctcga tgaaggcaac tttggcggag tcgagggctt
     3901 gggaggacat gatcaagttc ttgttttggt cgccgttgtc gacaagatcc aaaaaagaac
     3961 aattttcgtc gtagtgttat cgggcactgt ggtggttgtt ggaagatgga gtggaggcgg
     4021 acatcgccgt acgcgatatc gttcttgatt gcagccgaaa tccggcggct gcgagcatct
     4081 catcatgaat aatgcgcatg caaggaagaa ggtgccgtga tgagtattca aaatgaatga
     4141 tgatgatgct tgtctgacgt gtgtcttttc ttttacttct tcttcaaacg atttctgtta
     4201 ttgccctaga tcattttctt cctcacctct tcagaaacaa cactggttta taccaaaggt
     4261 aagcgaactc caatcatcat gtccttcttc gctttggaca ccgtggatca tgtcgatcgg
     4321 acaccggtta gcggggtgta ggtcgtcttc ttcatctagt cggtaatttg ccactgatga
     4381 tacccctcat aaggaacgca aacgtatatc acccatgcct atccgatgcg ctagtctcgc
     4441 aacaaaaaat ccacctctaa aaaatcgcgg tcgcagtaag cgatctttga acgcaaaata
     4501 cgaccagatt ttagggtctg tcaaaggtcg agcaacttcg ctctttgcaa agaaaccgaa
     4561 cagcgaacgt gggatccact agttctagag agcggactca cataagcatg caggattcga
     4621 gtggcatggt gtgcgctgta tgtatggttg caggaggaga ggatggtggt aataacaata
     4681 gcagcaacgt cactcgacgc gcgtctgggg tgccacaccg ggtaacgccg agtcgccgtc
     4741 agggtcgccg agccactctc acagcgtcac cgctggcacc cgctcagctt acagcttcta
     4801 tcctccgcca gcatccacat acatccccta taccgcatcc ccacccactg cccaaggtga
     4861 gtcatcttcc cgtcccctgc cccaccccgc cactcacctc tccaccatcc actactccgc
     4921 cgtattgcac ccaccgccta tcgcacatcc gagcacaagg gctggcctgc cacggctgct
     4981 aggtggcgct ctcccacacg ctgatctcca tgctggccat tggatcatgg gtgccaggtg
     5041 ctgggtgctg gatattggat gctggatgct gggtgcgcgc ttggtcgttt ccttccagga
     5101 ttgacggtcg ccgagaaagg acgacgtggc gcctgataac gagggccgac accaccgcat
     5161 cgcctcgacc tgcatccatc tcgccttgcg ctttgggcgc aacagcccat ccttgttggt
     5221 gctgtaagca tggctggaag agatggatgt gcgttggaca gagctgccgt cagggctttt
     5281 cggtgcacgg accctattgt cccccccaat cttccccgcg tatcctaata tgcgaccttt
     5341 tgctaattgt ctttttccat tagtaaaact cgcccaacac cggtgccacc atggactata
     5401 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
     5461 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt
     5521 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
     5581 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
     5641 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
     5701 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
     5761 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
     5821 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
     5881 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
     5941 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
     6001 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
     6061 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
     6121 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
     6181 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga
     6241 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
     6301 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
     6361 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
     6421 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
     6481 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
     6541 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
     6601 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
     6661 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
     6721 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
     6781 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
     6841 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
     6901 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
     6961 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
     7021 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
     7081 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
     7141 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
     7201 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
     7261 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
     7321 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
     7381 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
     7441 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
     7501 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
     7561 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
     7621 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
     7681 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     7741 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     7801 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     7861 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     7921 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     7981 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga
     8041 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     8101 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     8161 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     8221 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     8281 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     8341 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     8401 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     8461 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     8521 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     8581 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     8641 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     8701 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     8761 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     8821 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     8881 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     8941 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     9001 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     9061 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     9121 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     9181 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     9241 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     9301 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     9361 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     9421 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     9481 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     9541 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     9601 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt
     9661 aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     9721 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     9781 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     9841 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
     9901 gcggccgcca ccgcggtgga gctcccaatt cgccctatag tgagtcgtat tac
//
