LOCUS       sequence-233514-        8437 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses sgRNA targeting mouse Nanog and Cas9 nickase in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8437)
  AUTHORS   Ochiai H, Hayashi T, Umeda M, Yoshimura M, Harada A, Shimizu Y, 
            Nakano K, Saitoh N, Liu Z, Yamamoto T, Okamura T, Ohkawa Y, Kimura 
            H, Nikaido I
  TITLE     Genome-wide kinetic properties of transcriptional bursting in mouse 
            embryonic stem cells.
  JOURNAL   Sci Adv. 2020 Jun 17;6(25):eaaz6699. doi: 10.1126/sciadv.aaz6699. 
            eCollection 2020 Jun.
  PUBMED    32596448
REFERENCE   2  (bases 1 to 8437)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2020-06-17"; volume: "6(25):eaaz6699. doi"; pages: " 
            10.1126/sciadv.aaz6699. eCollection 2020 Jun"
COMMENT     Sequence Label: pCas9n-Nanog-R
FEATURES             Location/Qualifiers
     source          1..8437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        730..1007
                     /label=chicken beta-actin promoter
     intron          1008..1236
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1257..1283
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1287..1307
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1317..1340,1341..1343,1344..5417)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1317..5417
                     /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"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5421..5441
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5478..5495)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5484..5691
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5700..5840
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5700..5829
                     /label=AAV2 ITR
     rep_origin      5915..6370
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6002..6021)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6212..6233
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6387..6406)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6506..6528
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6566..6584)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6652..6756
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6757..6825,6826..7617)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6975..6994)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7788..8376
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8277..8296
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
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 caacatctgg gcttaaagtc gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
      901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga
     1021 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
     1081 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     1141 tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat
     1201 tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatgtacc
     1261 catacgatgt tccagattac gcttcgccga agaaaaagcg caaggtcgaa gcgtccgaca
     1321 agaagtacag catcggcctg gccatcggca ccaactctgt gggctgggcc gtgatcaccg
     1381 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
     1441 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc
     1501 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
     1561 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
     1621 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
     1681 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
     1741 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
     1801 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     1861 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     1921 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg
     1981 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcggc aacctgattg
     2041 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     2101 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     2161 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     2221 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     2281 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     2341 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     2401 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     2461 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc
     2521 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     2581 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     2641 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     2701 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     2761 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     2821 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg
     2881 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     2941 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     3001 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     3061 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     3121 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     3181 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     3241 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     3301 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     3361 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     3421 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     3481 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     3541 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     3601 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     3661 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     3721 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     3781 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc
     3841 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga
     3901 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     3961 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg
     4021 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     4081 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     4141 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     4201 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     4261 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     4321 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     4381 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     4441 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     4501 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg
     4561 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     4621 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa
     4681 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     4741 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     4801 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     4861 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     4921 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     4981 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     5041 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     5101 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     5161 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca
     5221 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     5281 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     5341 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     5401 ctcagctggg aggcgacagc cccaagaaga agagaaaggt ggaggccagc taagaattcc
     5461 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     5521 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     5581 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     5641 gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca
     5701 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     5761 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     5821 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     5881 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     5941 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     6001 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     6061 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     6121 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     6181 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     6241 ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     6301 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     6361 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6421 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     6481 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     6541 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     6601 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     6661 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     6721 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     6781 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     6841 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     6901 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6961 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     7021 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     7081 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     7141 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     7201 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     7261 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     7321 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     7381 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     7441 attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg
     7501 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     7561 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     7621 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     7681 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     7741 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     7801 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     7861 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     7921 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7981 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     8041 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     8101 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     8161 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     8221 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     8281 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     8341 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     8401 cggttcctgg ccttttgctg gccttttgct cacatgt
//
