LOCUS       PX330_PTEN        8507 bp DNA     circular SYN 23-SEP-2024
DEFINITION  A human codon-optimized SpCas9 and chimeric guide RNA expression 
            plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8507)
  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 Jan 3.
  PUBMED    23287718
REFERENCE   2  (bases 1 to 8507)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 23, 2024 from SnapGene 7.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2013 Jan 3."
FEATURES             Location/Qualifiers
     source          1..8507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..254
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     6..26
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
                     /note="color: black; sequence: GAGGGCCTATTTCCCATGATT"
     primer_bind     177..196
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
                     /note="color: black; sequence: GACTATCATATGCTTACCGT"
     misc_feature    255..275
                     /label=gRNA8 for PTEN KO
                     /note="gRNA8 for PTEN KO"
                     /note="color: #ff8000"
     misc_feature    255
                     /label=misc_feature 1
                     /note="color: #ff8000"
     misc_RNA        276..351
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     enhancer        448..733
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
                     /note="color: #ffffff"
     promoter        735..1011
                     /label=chicken beta-actin promoter
                     /note="color: #ffffff; direction: RIGHT"
     intron          1012..1239
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
                     /note="color: #------"
     regulatory      1251..1260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             1260..1325
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /note="color: #cc99b2
                     Cleavage site after base 1325"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1332..1352
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     CDS             1377..5477
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /note="color: #993366"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1377..5477
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /note="color: #993366"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5478..5525
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /note="color: #993366"
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5553..5570)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
                     /note="color: black; sequence: TAGAAGGCACAGTCGAGG"
     polyA_signal    5559..5766
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     repeat_region   5775..5915
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     repeat_region   5775..5904
                     /label=AAV2 ITR
                     /note="color: #ffcc99"
     rep_origin      5990..6445
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     primer_bind     complement(6077..6096)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="color: black; sequence: AGGGAAGAAAGCGAAAGGAG"
     primer_bind     6287..6308
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="color: black; sequence: GTGGACTCTTGTTCCAAACTGG"
     primer_bind     complement(6462..6481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /note="color: black; sequence: CGGCATCAGAGCAGATTGTA"
     primer_bind     6581..6603
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
                     /note="color: black; sequence: CCGGGAGCTGCATGTGTCAGAGG"
     primer_bind     complement(6641..6659)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
                     /note="color: black; sequence: AATAGGCGTATCACGAGGC"
     promoter        6727..6831
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             6832..7692
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 6832 .. 6900 / #ccffcc / signal sequence
                        2: 6901 .. 7692 / #ccffcc
                     Cleavage site after base 6900"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7050..7069)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: black; sequence: ATAATACCGCGCCACATAGC"
     rep_origin      7863..8451
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     8352..8371
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT"
ORIGIN
        1 catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
       61 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
      121 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
      181 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
      241 aaggacgaaa caccgtcacc tggattacag acccggtttt agagctagaa atagcaagtt
      301 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt
      361 ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt
      421 ctgcagacaa atggctctag aggtacccgt tacataactt acggtaaatg gcccgcctgg
      481 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca
      541 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      601 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg
      661 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      721 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
      781 ctcccccccc ccaattttgt atttatttat tttttaatta ttttgtgcgg gggggggggg
      841 gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg
      901 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
      961 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     1021 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     1081 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     1141 aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt
     1201 aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg
     1261 actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg
     1321 ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca
     1381 agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg
     1441 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
     1501 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc
     1561 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
     1621 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
     1681 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
     1741 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
     1801 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
     1861 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     1921 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     1981 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg
     2041 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg
     2101 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     2161 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     2221 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     2281 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     2341 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     2401 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     2461 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     2521 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc
     2581 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     2641 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     2701 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     2761 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     2821 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     2881 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg
     2941 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     3001 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     3061 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     3121 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     3181 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     3241 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     3301 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     3361 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     3421 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     3481 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     3541 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     3601 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     3661 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     3721 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     3781 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     3841 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc
     3901 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga
     3961 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     4021 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg
     4081 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     4141 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     4201 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     4261 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     4321 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     4381 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     4441 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     4501 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     4561 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg
     4621 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     4681 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa
     4741 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     4801 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     4861 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     4921 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     4981 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     5041 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     5101 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     5161 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     5221 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca
     5281 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     5341 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     5401 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     5461 ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga
     5521 aaaagtaaga attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     5581 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     5641 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     5701 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
     5761 tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     5821 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     5881 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     5941 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     6001 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     6061 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     6121 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     6181 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     6241 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     6301 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     6361 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     6421 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     6481 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     6541 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     6601 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     6661 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     6721 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     6781 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     6841 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     6901 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     6961 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     7021 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     7081 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     7141 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     7201 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     7261 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     7321 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     7381 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     7441 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     7501 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     7561 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     7621 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     7681 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     7741 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     7801 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7861 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7921 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7981 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     8041 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     8101 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     8161 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     8221 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     8281 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     8341 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     8401 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     8461 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctca
//
