LOCUS       px458_QC_intron_        9409 bp    DNA        circular     22-MAY-2020
DEFINITION  pSpCas9(BB)-2A-GFP (PX458).
ACCESSION   .
VERSION     .
KEYWORDS    px458
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9289)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 18 Feb 2016 from SnapGene Viewer 3.0.3
            http://www.snapgene.com
COMMENT     
COMMENT     
COMMENT     ApEinfo:methylated:1
FEATURES             Location/Qualifiers
     source          478..1657
                     /organism="pSpCas9(BB)-2A-GFP (PX458)"
                     /mol_type="other DNA"
                     /note="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /locus_tag="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /label="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /ApEinfo_label="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     source          473..477
                     /organism="pSpCas9(BB)-2A-GFP (PX458)"
                     /mol_type="other DNA"
                     /note="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /locus_tag="source:pSpCas9(BB)-2A-GFP (PX458)(1)"
                     /label="source:pSpCas9(BB)-2A-GFP (PX458)(1)"
                     /ApEinfo_label="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     source          1..245
                     /organism="pSpCas9(BB)-2A-GFP (PX458)"
                     /mol_type="other DNA"
                     /note="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /locus_tag="source:pSpCas9(BB)-2A-GFP (PX458)(2)"
                     /label="source:pSpCas9(BB)-2A-GFP (PX458)(2)"
                     /ApEinfo_label="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     source          431..436
                     /organism="pSpCas9(BB)-2A-GFP (PX458)"
                     /mol_type="other DNA"
                     /note="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /locus_tag="source:pSpCas9(BB)-2A-GFP (PX458)(3)"
                     /label="source:pSpCas9(BB)-2A-GFP (PX458)(3)"
                     /ApEinfo_label="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     promoter        1..241
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /locus_tag="U6 promoter"
                     /label="U6 promoter"
                     /ApEinfo_label="U6 promoter"
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     promoter        10..242
                     /note="hU6_promoter"
                     /locus_tag="hU6_promoter"
                     /label="hU6_promoter"
                     /ApEinfo_label="hU6_promoter"
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    172..191
                     /note="LKO1_5_primer"
                     /locus_tag="LKO1_5_primer"
                     /label="LKO1_5_primer"
                     /ApEinfo_label="LKO1_5_primer"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_RNA        271..346
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
                     /locus_tag="gRNA scaffold"
                     /label="gRNA scaffold"
                     /ApEinfo_label="gRNA scaffold"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     enhancer        478..763
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV
                     enhancer"
                     /locus_tag="CMV enhancer"
                     /label="CMV enhancer"
                     /ApEinfo_label="CMV enhancer"
                     /ApEinfo_fwdcolor="#5ac3fa"
                     /ApEinfo_revcolor="#5ac3fa"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    482..751
                     /note="CAG_enhancer"
                     /locus_tag="CAG_enhancer"
                     /label="CAG_enhancer"
                     /ApEinfo_label="CAG_enhancer"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     promoter        765..1042
                     /note="chicken beta-actin promoter"
                     /locus_tag="chicken beta-actin promoter"
                     /label="chicken beta-actin promoter"
                     /ApEinfo_label="chicken beta-actin promoter"
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     intron          1043..1271
                     /note="hybrid intron"
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
                     /locus_tag="hybrid intron"
                     /label="hybrid intron"
                     /ApEinfo_label="hybrid intron"
                     /ApEinfo_fwdcolor="#eb606c"
                     /ApEinfo_revcolor="#eb606c"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             complement(1268..1657)
                     /codon_start=1
                     /note="ORF frame 3"
                     /translation="MVHIVVGQPVDVQFLVHVHIPPILQVVQVQLLVLQLGVFHGVFF
                     Q
                     DLAAQLFDALFDPLHSLAAVLLSLLGGLVLSGHFDHDVLGLVPAHHFHELVHHLHCLQ
                     D
                     ALLNGGAAGQIGNVLVQAIALAGHLGFLDVLFKGQAVVVDQLHEVSVGEAVGLQEIQD
                     C
                     LAGLLVPDAVDQLPAQPAPAGVSPPLQLLHHFVVEQVGIGFQPFLDHLSVLKQCQGQH
                     D
                     IFQNVLVFLIVQEVLVLDNFQQIVVCAQGGVEPIFHAGDFHGVEALDFLEVVLFQLLH
                     G
                     HFPVGLEQQVHDGLFLLAAQEGGLSHSLGHVFHFGQLVIHGEVLVQQAVLGQHLLVGQ
                     V
                     LIEVGHPLDEALGGSALVHHFLEVPGGDGFLALSGHPGESAVSPGQRAHVVGDAEGQD
                     L
                     LDLFPVVLQEWVKIFLPPQNGVQLSQVDLVGDAAVVEGPLLPQQVLSVQLHEQFLGAV
                     H
                     LFQDGLDELVELFLAGSAVNVAGVAVLALVEENLFVLLRQLLPHESFQQGQVLVVLVV
                     S
                     LDHRGAQGGLGDLGVHSQDVAQQDGVGQVLGGQKQVGVLVADLGQQVVQVVVVGVLAQ
                     L
                     QFGILGQVEVALEVGGQAQAQGNQVSEQAILLLAGQLGDQIFQPSALAQSGRQDGLGV
                     H
                     AAGVDGVFLEQLVVGLHQLDEQLVHVAVVGVQVALDQEVAPELDHVGQGQIDQPQVGL
                     V
                     GAVHQFLSQVVDGGVLLVVGHLVHDVAEDGVPLVLLILFHQEGLFQSVEEAVVHLGHL
                     V
                     AEDLLQIADPVLPSGVSSSGGSLQPGGLGCFAAVEQQGSDQVLLDAVPVGVAQHLEFL
                     A
                     GHLVLVGDHGPAHRVGADVQADAVLLVGCWDSVDTDLPLLLWGHLIVIVFVINIMILV
                     V SVVVLIVHGGTGPT"
                     /locus_tag="ORF frame 3"
                     /label="ORF frame 3"
                     /ApEinfo_label="ORF frame 3"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             1289..1657
                     /codon_start=1
                     /note="ORF frame 3(1)"
                     /translation="MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKY
                     S
                     IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL
                     K
                     RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV
                     D
                     EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD
                     K
                     LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL
                     I
                     ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
                     L
                     LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY
                     A
                     GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE
                     L
                     HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN
                     F
                     EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK
                     P
                     AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH
                     D
                     LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR
                     R
                     YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV
                     S
                     GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK
                     G
                     QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN
                     R
                     LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK
                     L
                     ITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL
                     I
                     REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF
                     V
                     YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG
                     E
                     TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW
                     D
                     PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
                     G
                     YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEK
                     L
                     KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR
                     E
                     QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL
                     S
                     QLGGDKRPAATKKAGQAKKKKEFGSGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPI
                     L
                     VELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR
                     Y
                     PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE
                     D
                     GNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDG
                     P VLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKEF"
                     /locus_tag="ORF frame 3(1)"
                     /label="ORF frame 3(1)"
                     /ApEinfo_label="ORF frame 3(1)"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             1292..1357
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by
                     an enterokinase cleavage site"
                     /note="3xFLAG"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /locus_tag="three tandem FLAG(R) epitope tags, followed by
                     an enterokinase cleavage site"
                     /label="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /ApEinfo_label="three tandem FLAG(R) epitope tags,
                     followed by an enterokinase cleavage site"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    1292..1312
                     /note="FLAG"
                     /locus_tag="FLAG"
                     /label="FLAG"
                     /ApEinfo_label="FLAG"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    1313..1333
                     /note="FLAG(1)"
                     /locus_tag="FLAG(1)"
                     /label="FLAG(1)"
                     /ApEinfo_label="FLAG(1)"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    1334..1357
                     /note="FLAG(2)"
                     /locus_tag="FLAG(2)"
                     /label="FLAG(2)"
                     /ApEinfo_label="FLAG(2)"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    1364..1387
                     /note="SV40 NLS"
                     /locus_tag="SV40 NLS"
                     /label="SV40 NLS"
                     /ApEinfo_label="SV40 NLS"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             1364..1384
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
                     /locus_tag="nuclear localization signal of SV40 large T
                     antigen"
                     /label="nuclear localization signal of SV40 large T
                     antigen"
                     /ApEinfo_label="nuclear localization signal of SV40 large
                     T antigen"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    1364..1384
                     /note="SV40 NLS(1)"
                     /locus_tag="SV40 NLS(1)"
                     /label="SV40 NLS(1)"
                     /ApEinfo_label="SV40 NLS(1)"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    1370..1384
                     /note="NLS"
                     /locus_tag="NLS"
                     /label="NLS"
                     /ApEinfo_label="NLS"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             1409..1657
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /note="Cas9"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     N
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     F
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     F
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     E
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     I
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     Q
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     R
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     N
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     F
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     S
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     L
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     Q
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     H
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     L
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     S
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     V
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     N
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     E
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     E
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     T
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     L
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     A
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     D ATLIHQSITGLYETRIDLSQLGGD"
                     /locus_tag="Cas9 (Csn1) endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /ApEinfo_label="Cas9 (Csn1) endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             5592..5639
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
                     /locus_tag="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /ApEinfo_label="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             5655..5708
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid
                     protein"
                     /note="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
                     /locus_tag="2A peptide from Thosea asigna virus capsid
                     protein"
                     /label="2A peptide from Thosea asigna virus capsid
                     protein"
                     /ApEinfo_label="2A peptide from Thosea asigna virus capsid
                     protein"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             5709..6422
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="EGFP"
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     K
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     G
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     V
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     E FVTAAGITLGMDELYK"
                     /locus_tag="enhanced GFP"
                     /label="enhanced GFP"
                     /ApEinfo_label="enhanced GFP"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     gene            5709..6422
                     /gene="EGFP"
                     /note="EGFP"
                     /locus_tag="EGFP"
                     /label="EGFP"
                     /ApEinfo_label="EGFP"
                     /ApEinfo_fwdcolor="#ff797d"
                     /ApEinfo_revcolor="#ff102f"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    5709..6422
                     /note="EGFP(1)"
                     /locus_tag="EGFP(1)"
                     /label="EGFP(1)"
                     /ApEinfo_label="EGFP(1)"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(5751..5772)
                     /note="EGFP_N_primer"
                     /locus_tag="EGFP_N_primer"
                     /label="EGFP_N_primer"
                     /ApEinfo_label="EGFP_N_primer"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    5889..5918
                     /note="Y66 (EGFP)"
                     /locus_tag="Y66 (EGFP)"
                     /label="Y66 (EGFP)"
                     /ApEinfo_label="Y66 (EGFP)"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    6359..6380
                     /note="EGFP_C_primer"
                     /locus_tag="EGFP_C_primer"
                     /label="EGFP_C_primer"
                     /ApEinfo_label="EGFP_C_primer"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(6450..6467)
                     /note="BGH_rev_primer"
                     /locus_tag="BGH_rev_primer"
                     /label="BGH_rev_primer"
                     /ApEinfo_label="BGH_rev_primer"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     terminator      6453..6656
                     /note="bGH_PA_terminator"
                     /locus_tag="bGH_PA_terminator"
                     /label="bGH_PA_terminator"
                     /ApEinfo_label="bGH_PA_terminator"
                     /ApEinfo_fwdcolor="#9d1b1c"
                     /ApEinfo_revcolor="#9d1b1c"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     polyA_signal    6456..6663
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
                     /locus_tag="bGH poly(A) signal"
                     /label="bGH poly(A) signal"
                     /ApEinfo_label="bGH poly(A) signal"
                     /ApEinfo_fwdcolor="#ff3eee"
                     /ApEinfo_revcolor="#ff3eee"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     repeat_region   6672..6812
                     /note="AAV2 ITR"
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /locus_tag="AAV2 ITR"
                     /label="AAV2 ITR"
                     /ApEinfo_label="AAV2 ITR"
                     /ApEinfo_fwdcolor="#0dfff7"
                     /ApEinfo_revcolor="#0dfff7"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     rep_origin      6887..7342
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /locus_tag="f1 ori"
                     /label="f1 ori"
                     /ApEinfo_label="f1 ori"
                     /ApEinfo_fwdcolor="#999999"
                     /ApEinfo_revcolor="#999999"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     rep_origin      6904..7210
                     /note="f1_origin"
                     /locus_tag="f1_origin"
                     /label="f1_origin"
                     /ApEinfo_label="f1_origin"
                     /ApEinfo_fwdcolor="#999999"
                     /ApEinfo_revcolor="#999999"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    7478..7500
                     /note="pGEX_3_primer"
                     /locus_tag="pGEX_3_primer"
                     /label="pGEX_3_primer"
                     /ApEinfo_label="pGEX_3_primer"
                     /ApEinfo_fwdcolor="#7eff74"
                     /ApEinfo_revcolor="#7eff74"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     promoter        7624..7728
                     /gene="bla"
                     /note="AmpR promoter"
                     /locus_tag="bla"
                     /label="bla"
                     /ApEinfo_label="bla"
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     promoter        7659..7687
                     /note="AmpR_promoter"
                     /locus_tag="AmpR_promoter"
                     /label="AmpR_promoter"
                     /ApEinfo_label="AmpR_promoter"
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     gene            7729..8589
                     /gene="Ampicillin"
                     /note="Ampicillin"
                     /locus_tag="Ampicillin"
                     /label="Ampicillin"
                     /ApEinfo_label="Ampicillin"
                     /ApEinfo_fwdcolor="#ff797d"
                     /ApEinfo_revcolor="#ff102f"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             7729..8589
                     /codon_start=1
                     /note="ORF frame 1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     I
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     S
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     W
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     A
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     S LIKHW"
                     /locus_tag="ORF frame 1"
                     /label="ORF frame 1"
                     /ApEinfo_label="ORF frame 1"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     rep_origin      8744..9363
                     /note="pBR322_origin"
                     /locus_tag="pBR322_origin"
                     /label="pBR322_origin"
                     /ApEinfo_label="pBR322_origin"
                     /ApEinfo_fwdcolor="#999999"
                     /ApEinfo_revcolor="#999999"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     rep_origin      8760..9348
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /locus_tag="ori"
                     /label="ori"
                     /ApEinfo_label="ori"
                     /ApEinfo_fwdcolor="#999999"
                     /ApEinfo_revcolor="#999999"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     source          1658..3259
                     /organism="pSpCas9(BB)-2A-GFP (PX458)"
                     /mol_type="other DNA"
                     /note="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /locus_tag="source:pSpCas9(BB)-2A-GFP (PX458)(4)"
                     /label="source:pSpCas9(BB)-2A-GFP (PX458)(4)"
                     /ApEinfo_label="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             complement(1658..3259)
                     /codon_start=1
                     /note="ORF frame 3"
                     /translation="MVHIVVGQPVDVQFLVHVHIPPILQVVQVQLLVLQLGVFHGVFF
                     Q
                     DLAAQLFDALFDPLHSLAAVLLSLLGGLVLSGHFDHDVLGLVPAHHFHELVHHLHCLQ
                     D
                     ALLNGGAAGQIGNVLVQAIALAGHLGFLDVLFKGQAVVVDQLHEVSVGEAVGLQEIQD
                     C
                     LAGLLVPDAVDQLPAQPAPAGVSPPLQLLHHFVVEQVGIGFQPFLDHLSVLKQCQGQH
                     D
                     IFQNVLVFLIVQEVLVLDNFQQIVVCAQGGVEPIFHAGDFHGVEALDFLEVVLFQLLH
                     G
                     HFPVGLEQQVHDGLFLLAAQEGGLSHSLGHVFHFGQLVIHGEVLVQQAVLGQHLLVGQ
                     V
                     LIEVGHPLDEALGGSALVHHFLEVPGGDGFLALSGHPGESAVSPGQRAHVVGDAEGQD
                     L
                     LDLFPVVLQEWVKIFLPPQNGVQLSQVDLVGDAAVVEGPLLPQQVLSVQLHEQFLGAV
                     H
                     LFQDGLDELVELFLAGSAVNVAGVAVLALVEENLFVLLRQLLPHESFQQGQVLVVLVV
                     S
                     LDHRGAQGGLGDLGVHSQDVAQQDGVGQVLGGQKQVGVLVADLGQQVVQVVVVGVLAQ
                     L
                     QFGILGQVEVALEVGGQAQAQGNQVSEQAILLLAGQLGDQIFQPSALAQSGRQDGLGV
                     H
                     AAGVDGVFLEQLVVGLHQLDEQLVHVAVVGVQVALDQEVAPELDHVGQGQIDQPQVGL
                     V
                     GAVHQFLSQVVDGGVLLVVGHLVHDVAEDGVPLVLLILFHQEGLFQSVEEAVVHLGHL
                     V
                     AEDLLQIADPVLPSGVSSSGGSLQPGGLGCFAAVEQQGSDQVLLDAVPVGVAQHLEFL
                     A
                     GHLVLVGDHGPAHRVGADVQADAVLLVGCWDSVDTDLPLLLWGHLIVIVFVINIMILV
                     V SVVVLIVHGGTGPT"
                     /locus_tag="ORF frame 3(2)"
                     /label="ORF frame 3(2)"
                     /ApEinfo_label="ORF frame 3"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             1658..3259
                     /codon_start=1
                     /note="ORF frame 3(1)"
                     /translation="MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKY
                     S
                     IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL
                     K
                     RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV
                     D
                     EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD
                     K
                     LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL
                     I
                     ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
                     L
                     LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY
                     A
                     GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE
                     L
                     HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN
                     F
                     EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK
                     P
                     AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH
                     D
                     LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR
                     R
                     YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV
                     S
                     GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK
                     G
                     QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN
                     R
                     LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK
                     L
                     ITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL
                     I
                     REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF
                     V
                     YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG
                     E
                     TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW
                     D
                     PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
                     G
                     YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEK
                     L
                     KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR
                     E
                     QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL
                     S
                     QLGGDKRPAATKKAGQAKKKKEFGSGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPI
                     L
                     VELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR
                     Y
                     PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE
                     D
                     GNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDG
                     P VLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKEF"
                     /locus_tag="ORF frame 3(1)(1)"
                     /label="ORF frame 3(1)(1)"
                     /ApEinfo_label="ORF frame 3(1)"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             1658..3259
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /note="Cas9"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     N
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     F
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     F
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     E
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     I
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     Q
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     R
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     N
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     F
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     S
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     L
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     Q
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     H
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     L
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     S
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     V
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     N
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     E
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     E
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     T
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     L
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     A
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     D ATLIHQSITGLYETRIDLSQLGGD"
                     /locus_tag="Cas9 (Csn1) endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system(1)"
                     /label="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system(1)"
                     /ApEinfo_label="Cas9 (Csn1) endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     source          3260..3541
                     /organism="pSpCas9(BB)-2A-GFP (PX458)"
                     /mol_type="other DNA"
                     /note="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /locus_tag="source:pSpCas9(BB)-2A-GFP (PX458)(5)"
                     /label="source:pSpCas9(BB)-2A-GFP (PX458)(5)"
                     /ApEinfo_label="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             complement(3260..3541)
                     /codon_start=1
                     /note="ORF frame 3"
                     /translation="MVHIVVGQPVDVQFLVHVHIPPILQVVQVQLLVLQLGVFHGVFF
                     Q
                     DLAAQLFDALFDPLHSLAAVLLSLLGGLVLSGHFDHDVLGLVPAHHFHELVHHLHCLQ
                     D
                     ALLNGGAAGQIGNVLVQAIALAGHLGFLDVLFKGQAVVVDQLHEVSVGEAVGLQEIQD
                     C
                     LAGLLVPDAVDQLPAQPAPAGVSPPLQLLHHFVVEQVGIGFQPFLDHLSVLKQCQGQH
                     D
                     IFQNVLVFLIVQEVLVLDNFQQIVVCAQGGVEPIFHAGDFHGVEALDFLEVVLFQLLH
                     G
                     HFPVGLEQQVHDGLFLLAAQEGGLSHSLGHVFHFGQLVIHGEVLVQQAVLGQHLLVGQ
                     V
                     LIEVGHPLDEALGGSALVHHFLEVPGGDGFLALSGHPGESAVSPGQRAHVVGDAEGQD
                     L
                     LDLFPVVLQEWVKIFLPPQNGVQLSQVDLVGDAAVVEGPLLPQQVLSVQLHEQFLGAV
                     H
                     LFQDGLDELVELFLAGSAVNVAGVAVLALVEENLFVLLRQLLPHESFQQGQVLVVLVV
                     S
                     LDHRGAQGGLGDLGVHSQDVAQQDGVGQVLGGQKQVGVLVADLGQQVVQVVVVGVLAQ
                     L
                     QFGILGQVEVALEVGGQAQAQGNQVSEQAILLLAGQLGDQIFQPSALAQSGRQDGLGV
                     H
                     AAGVDGVFLEQLVVGLHQLDEQLVHVAVVGVQVALDQEVAPELDHVGQGQIDQPQVGL
                     V
                     GAVHQFLSQVVDGGVLLVVGHLVHDVAEDGVPLVLLILFHQEGLFQSVEEAVVHLGHL
                     V
                     AEDLLQIADPVLPSGVSSSGGSLQPGGLGCFAAVEQQGSDQVLLDAVPVGVAQHLEFL
                     A
                     GHLVLVGDHGPAHRVGADVQADAVLLVGCWDSVDTDLPLLLWGHLIVIVFVINIMILV
                     V SVVVLIVHGGTGPT"
                     /locus_tag="ORF frame 3(3)"
                     /label="ORF frame 3(3)"
                     /ApEinfo_label="ORF frame 3"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             3260..3541
                     /codon_start=1
                     /note="ORF frame 3(1)"
                     /translation="MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKY
                     S
                     IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL
                     K
                     RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV
                     D
                     EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD
                     K
                     LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL
                     I
                     ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
                     L
                     LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY
                     A
                     GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE
                     L
                     HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN
                     F
                     EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK
                     P
                     AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH
                     D
                     LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR
                     R
                     YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV
                     S
                     GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK
                     G
                     QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN
                     R
                     LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK
                     L
                     ITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL
                     I
                     REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF
                     V
                     YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG
                     E
                     TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW
                     D
                     PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
                     G
                     YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEK
                     L
                     KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR
                     E
                     QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL
                     S
                     QLGGDKRPAATKKAGQAKKKKEFGSGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPI
                     L
                     VELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR
                     Y
                     PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE
                     D
                     GNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDG
                     P VLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKEF"
                     /locus_tag="ORF frame 3(1)(2)"
                     /label="ORF frame 3(1)(2)"
                     /ApEinfo_label="ORF frame 3(1)"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             3260..3541
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /note="Cas9"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     N
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     F
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     F
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     E
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     I
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     Q
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     R
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     N
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     F
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     S
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     L
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     Q
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     H
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     L
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     S
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     V
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     N
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     E
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     E
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     T
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     L
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     A
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     D ATLIHQSITGLYETRIDLSQLGGD"
                     /locus_tag="Cas9 (Csn1) endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system(2)"
                     /label="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system(2)"
                     /ApEinfo_label="Cas9 (Csn1) endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     source          3624..9409
                     /organism="pSpCas9(BB)-2A-GFP (PX458)"
                     /mol_type="other DNA"
                     /note="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /locus_tag="source:pSpCas9(BB)-2A-GFP (PX458)(6)"
                     /label="source:pSpCas9(BB)-2A-GFP (PX458)(6)"
                     /ApEinfo_label="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             complement(3624..4007)
                     /codon_start=1
                     /note="ORF frame 3"
                     /translation="MVHIVVGQPVDVQFLVHVHIPPILQVVQVQLLVLQLGVFHGVFF
                     Q
                     DLAAQLFDALFDPLHSLAAVLLSLLGGLVLSGHFDHDVLGLVPAHHFHELVHHLHCLQ
                     D
                     ALLNGGAAGQIGNVLVQAIALAGHLGFLDVLFKGQAVVVDQLHEVSVGEAVGLQEIQD
                     C
                     LAGLLVPDAVDQLPAQPAPAGVSPPLQLLHHFVVEQVGIGFQPFLDHLSVLKQCQGQH
                     D
                     IFQNVLVFLIVQEVLVLDNFQQIVVCAQGGVEPIFHAGDFHGVEALDFLEVVLFQLLH
                     G
                     HFPVGLEQQVHDGLFLLAAQEGGLSHSLGHVFHFGQLVIHGEVLVQQAVLGQHLLVGQ
                     V
                     LIEVGHPLDEALGGSALVHHFLEVPGGDGFLALSGHPGESAVSPGQRAHVVGDAEGQD
                     L
                     LDLFPVVLQEWVKIFLPPQNGVQLSQVDLVGDAAVVEGPLLPQQVLSVQLHEQFLGAV
                     H
                     LFQDGLDELVELFLAGSAVNVAGVAVLALVEENLFVLLRQLLPHESFQQGQVLVVLVV
                     S
                     LDHRGAQGGLGDLGVHSQDVAQQDGVGQVLGGQKQVGVLVADLGQQVVQVVVVGVLAQ
                     L
                     QFGILGQVEVALEVGGQAQAQGNQVSEQAILLLAGQLGDQIFQPSALAQSGRQDGLGV
                     H
                     AAGVDGVFLEQLVVGLHQLDEQLVHVAVVGVQVALDQEVAPELDHVGQGQIDQPQVGL
                     V
                     GAVHQFLSQVVDGGVLLVVGHLVHDVAEDGVPLVLLILFHQEGLFQSVEEAVVHLGHL
                     V
                     AEDLLQIADPVLPSGVSSSGGSLQPGGLGCFAAVEQQGSDQVLLDAVPVGVAQHLEFL
                     A
                     GHLVLVGDHGPAHRVGADVQADAVLLVGCWDSVDTDLPLLLWGHLIVIVFVINIMILV
                     V SVVVLIVHGGTGPT"
                     /locus_tag="ORF frame 3(4)"
                     /label="ORF frame 3(4)"
                     /ApEinfo_label="ORF frame 3"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             3624..6431
                     /codon_start=1
                     /note="ORF frame 3(1)"
                     /translation="MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKY
                     S
                     IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL
                     K
                     RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV
                     D
                     EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD
                     K
                     LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL
                     I
                     ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
                     L
                     LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY
                     A
                     GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE
                     L
                     HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN
                     F
                     EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK
                     P
                     AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH
                     D
                     LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR
                     R
                     YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV
                     S
                     GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK
                     G
                     QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN
                     R
                     LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK
                     L
                     ITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL
                     I
                     REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF
                     V
                     YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG
                     E
                     TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW
                     D
                     PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
                     G
                     YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEK
                     L
                     KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR
                     E
                     QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL
                     S
                     QLGGDKRPAATKKAGQAKKKKEFGSGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPI
                     L
                     VELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR
                     Y
                     PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE
                     D
                     GNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDG
                     P VLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKEF"
                     /locus_tag="ORF frame 3(1)(3)"
                     /label="ORF frame 3(1)(3)"
                     /ApEinfo_label="ORF frame 3(1)"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             3624..5591
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /note="Cas9"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     N
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     F
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     F
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     E
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     I
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     Q
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     R
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     N
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     F
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     S
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     L
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     Q
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     H
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     L
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     S
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     V
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     N
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     E
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     E
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     T
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     L
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     A
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     D ATLIHQSITGLYETRIDLSQLGGD"
                     /locus_tag="Cas9 (Csn1) endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system(3)"
                     /label="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system(3)"
                     /ApEinfo_label="Cas9 (Csn1) endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     source          271..430
                     /organism="pSpCas9(BB)-2A-GFP (PX458)"
                     /mol_type="other DNA"
                     /note="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /locus_tag="source:pSpCas9(BB)-2A-GFP (PX458)(7)"
                     /label="source:pSpCas9(BB)-2A-GFP (PX458)(7)"
                     /ApEinfo_label="source:pSpCas9(BB)-2A-GFP (PX458)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaaCACCG TCGGCTTCCC ATTCTCCGGT gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaacgc gtAGGCCTtG AGACCtagGG TCTCtgctag cggtacccgt
      481 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      541 gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      601 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      661 caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat gggactttcc
      721 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
      781 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
      841 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg
      901 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca
      961 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
     1021 aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc gtgccccgct
     1081 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     1141 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
     1201 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
     1261 ctttttttca ggttggACCG GTgccaccat ggactataag gaccacgacg gagactacaa
     1321 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
     1381 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg
     1441 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     1501 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     1561 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     1621 caccagacgg aagaaccgga tctgctatct gcaagAGATC Ttcagcaacg agatggccaa
     1681 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     1741 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     1801 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     1861 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     1921 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     1981 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     2041 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     2101 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     2161 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     2221 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     2281 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     2341 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     2401 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     2461 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     2521 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     2581 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     2641 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     2701 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     2761 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     2821 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     2881 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     2941 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     3001 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     3061 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     3121 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     3181 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     3241 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     3301 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     3361 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     3421 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     3481 cttcatgcag ctgatCCACG ACGACAGCCT GACCTTTAAA GAGGACATCC AGAAAGCCCA
     3541 GGTAAGGGGC TCACAGTAGC AGGCTTGAGG TCTGGACATA TATATGGGTG ACAATGACAT
     3601 CCACTTTGCC TTTCTCTCCA CAGgtgtccg gccagggcga tagcctgcac gagcacattg
     3661 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     3721 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     3781 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     3841 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     3901 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     3961 accaggaact ggacatcaac cggctgtccG ACtacgatgt ggaccatatc gtgcctcaga
     4021 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     4081 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     4141 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     4201 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     4261 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     4321 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     4381 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     4441 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     4501 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     4561 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     4621 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     4681 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     4741 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     4801 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     4861 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     4921 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     4981 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     5041 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     5101 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     5161 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     5221 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     5281 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     5341 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     5401 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     5461 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     5521 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     5581 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     5641 aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc
     5701 ctggcccagt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     5761 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     5821 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     5881 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     5941 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     6001 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     6061 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     6121 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     6181 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     6241 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     6301 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     6361 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     6421 aggaattcta actagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     6481 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     6541 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     6601 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg
     6661 ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     6721 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     6781 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     6841 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     6901 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     6961 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     7021 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     7081 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     7141 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     7201 actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg
     7261 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     7321 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     7381 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     7441 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     7501 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     7561 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     7621 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     7681 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     7741 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     7801 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     7861 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     7921 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     7981 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     8041 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     8101 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     8161 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     8221 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     8281 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     8341 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     8401 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt
     8461 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     8521 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     8581 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     8641 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     8701 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     8761 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     8821 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     8881 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     8941 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     9001 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     9061 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     9121 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     9181 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     9241 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     9301 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     9361 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt 
//
