LOCUS       pX330        8504 bp DNA     circular SYN 20-JUL-2025
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 8504)
  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 8504)
  AUTHORS   Zhang Lab / Addgene #42230
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8504)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8504)
  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     SGRef: number: 3; 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..8504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          51..70
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          207..434
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             452..454
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             455..520
                     /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             527..547
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /label=nuclear localization signal of SV40 large T
                     ant
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             572..4672
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             4673..4720
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=bipartite nuclear localization signal from
                     nucl
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    4754..4961
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4970..5110
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5185..5640
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5922..6026
                     /label=AmpR promoter
     CDS             6027..6884
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      7058..7646
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        7708..7948
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        7975..8050
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      8051..8056
                     /note="pol III terminator"
                     /note="RNA polymerase III transcription terminator"
     enhancer        8147..8432
                     /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 cgcgcgccag
       61 gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
      121 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
      181 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc
      241 gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg
      301 tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt
      361 ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa
      421 tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta
      481 caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg
      541 gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat
      601 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
      661 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
      721 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
      781 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
      841 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
      901 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
      961 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     1021 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     1081 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     1141 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     1201 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     1261 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     1321 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     1381 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     1441 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     1501 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     1561 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     1621 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     1681 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     1741 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     1801 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     1861 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     1921 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     1981 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     2041 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     2101 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     2161 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     2221 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     2281 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     2341 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     2401 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     2461 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     2521 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     2581 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     2641 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     2701 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     2761 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     2821 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     2881 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     2941 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     3001 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     3061 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     3121 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     3181 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     3241 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     3301 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     3361 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     3421 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     3481 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     3541 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     3601 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     3661 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     3721 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     3781 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     3841 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     3901 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     3961 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     4021 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     4081 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     4141 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     4201 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     4261 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     4321 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     4381 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     4441 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     4501 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     4561 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     4621 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc
     4681 ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag taagaattcc tagagctcgc
     4741 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4801 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4861 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4921 aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta
     4981 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     5041 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     5101 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5161 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5221 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     5281 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5341 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5401 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5461 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     5521 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     5581 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     5641 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     5701 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5761 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     5821 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5881 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5941 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     6001 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     6061 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6121 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6181 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6241 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6301 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6361 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6421 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6481 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6541 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6601 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6661 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6721 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     6781 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6841 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6901 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6961 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     7021 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     7081 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     7141 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7201 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7261 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     7321 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     7381 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7441 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7501 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7561 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7621 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7681 ccttttgctg gccttttgct cacatgtgag ggcctatttc ccatgattcc ttcatatttg
     7741 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     7801 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     7861 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     7921 ttggctttat atatcttgtg gaaaggacga aacaccgggt cttcgagaag acctgtttta
     7981 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     8041 gagtcggtgc ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     8101 tttagcgcgt gcgccaattc tgcagacaaa tggctctaga ggtacccgtt acataactta
     8161 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa
     8221 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     8281 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     8341 aatggcccgc ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt
     8401 acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca
     8461 ctctccccat ctcccccccc tccccacccc caattttgta ttta
//
