LOCUS       pX330.        8484 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Zhang lab plasmid for expressing a chimeric guide RNA (gRNA) 
            together with human codon-optimized Cas9. Also known as 
            pX330-U6-Chimeric_BB-CBh-hSpCas9.
ACCESSION   .
VERSION     .
KEYWORDS    pX330
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8484)
  AUTHORS   Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, 
            Jiang W, Marraffini LA, Zhang F.
  TITLE     Multiplex genome engineering using CRISPR/Cas systems.
  JOURNAL   Science 2013;339:819-23.
  PUBMED    23287718
REFERENCE   2  (bases 1 to 8484)
  AUTHORS   Zhang Lab / Addgene #42230
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8484)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science"; 
            date: "2013"; volume: "339"; pages: "819-23"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     Digest with BbsI to insert annealed oligos encoding the guide 
            sequence.
            NGS sequence provided by Addgene.
FEATURES             Location/Qualifiers
     source          1..8484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          187..414
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             432..434
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             435..500
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=three tandem FLAG
                     /note="3xFLAG"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             507..527
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             552..4652
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             4653..4700
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=bipartite nuclear localization signal from
                     nucl
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    4734..4941
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4950..5090
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5165..5620
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5902..6006
                     /label=AmpR promoter
     CDS             6007..6864
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      7038..7626
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        7688..7928
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        7955..8030
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      8031..8036
                     /note="pol III terminator"
                     /note="RNA polymerase III transcription terminator"
     enhancer        8127..8412
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg gcggggcgag
       61 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
      121 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
      181 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
      241 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
      301 tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt
      361 ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga
      421 ccggtgccac catggactat aaggaccacg acggagacta caaggatcat gatattgatt
      481 acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag
      541 tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct
      601 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
      661 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa
      721 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
      781 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
      841 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
      901 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
      961 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
     1021 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     1081 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     1141 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     1201 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     1261 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     1321 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     1381 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     1441 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     1501 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     1561 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     1621 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     1681 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     1741 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     1801 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     1861 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     1921 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     1981 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact
     2041 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     2101 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     2161 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga
     2221 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     2281 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     2341 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     2401 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     2461 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     2521 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
     2581 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
     2641 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     2701 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     2761 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     2821 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     2881 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     2941 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     3001 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     3061 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
     3121 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     3181 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
     3241 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     3301 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     3361 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
     3421 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     3481 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
     3541 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     3601 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     3661 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     3721 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
     3781 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
     3841 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
     3901 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     3961 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     4021 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     4081 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     4141 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     4201 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     4261 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     4321 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     4381 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     4441 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     4501 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     4561 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     4621 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg
     4681 gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc
     4741 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4801 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4861 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4921 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     4981 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     5041 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     5101 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     5161 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5221 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     5281 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5341 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     5401 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5461 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     5521 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     5581 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     5641 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5701 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5761 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5821 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5881 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5941 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     6001 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6061 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     6121 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     6181 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     6241 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     6301 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     6361 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6421 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6481 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6541 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6601 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6661 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6721 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6781 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6841 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6901 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6961 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     7021 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     7081 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     7141 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     7201 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     7261 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     7321 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     7381 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7441 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7501 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7561 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7621 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7681 cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     7741 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     7801 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     7861 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     7921 gaaaggacga aacaccgggt cttcgagaag acctgtttta gagctagaaa tagcaagtta
     7981 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt
     8041 tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt gcgccaattc
     8101 tgcagacaaa tggctctaga ggtacccgtt acataactta cggtaaatgg cccgcctggc
     8161 tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg gactttccat
     8221 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     8281 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattgt
     8341 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     8401 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     8461 tccccacccc caattttgta ttta
//
