LOCUS       sequence-238824-        8445 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting mouse Eif4a1 and Cas9 nickase in mammalian
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8445)
  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 8445)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 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: px335-sgRNA-EF(Cas9n)-Eif4a1-L
FEATURES             Location/Qualifiers
     source          1..8445
                     /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"
     enhancer        453..738
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1017..1244
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1265..1291
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1295..1315
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1325..1348,1349..1351,1352..5425)
                     /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             1325..5425
                     /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             5429..5449
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5486..5503)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5492..5699
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5708..5848
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5708..5837
                     /label=AAV2 ITR
     rep_origin      5923..6378
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6010..6029)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6220..6241
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6395..6414)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6514..6536
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6574..6592)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6660..6764
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6765..6833,6834..7625)
                     /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(6983..7002)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7796..8384
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8285..8304
                     /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 atgatcctta gaatctaggg gtttaagagc tatgctggaa acagcatagc
      301 aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      361 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc
      421 caattctgca gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg
      481 cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact
      541 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      601 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      661 cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      721 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
      781 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
      841 ggggcggggg gggggggggg cgcgcgccag gcgggggggg gggggggggg gggggggggg
      901 gggggggggg ggggggggcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
      961 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
     1021 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
     1081 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
     1141 gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa
     1201 tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac
     1261 catgtaccca tacgatgttc cagattacgc ttcgccgaag aaaaagcgca aggtcgaagc
     1321 gtccgacaag aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt
     1381 gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg
     1441 gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga
     1501 ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg
     1561 ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag
     1621 actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg
     1681 caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa
     1741 gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca
     1801 catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga
     1861 cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc
     1921 catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag
     1981 acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggcaa
     2041 cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga
     2101 ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc
     2161 ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat
     2221 cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc
     2281 tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg
     2341 gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc
     2401 cggctacatt gacggcggag ccagccagga agagttctac aagttcatca agcccatcct
     2461 ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg
     2521 gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca
     2581 cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat
     2641 cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag
     2701 cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga
     2761 agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa
     2821 gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt
     2881 gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct
     2941 gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac
     3001 cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat
     3061 ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat
     3121 tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt
     3181 gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc
     3241 ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg
     3301 caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct
     3361 ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga
     3421 cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct
     3481 gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac
     3541 agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt
     3601 gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag
     3661 aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc
     3721 cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg
     3781 ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca
     3841 tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag
     3901 cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa
     3961 gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct
     4021 gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca
     4081 gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa
     4141 cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc
     4201 caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa
     4261 ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa
     4321 gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa
     4381 gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag
     4441 caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg
     4501 gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt
     4561 tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt
     4621 gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat
     4681 cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc
     4741 ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt
     4801 gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga
     4861 ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa
     4921 gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact
     4981 gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag
     5041 ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga
     5101 acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt
     5161 gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa
     5221 gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc
     5281 ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa
     5341 agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat
     5401 cgacctgtct cagctgggag gcgacagccc caagaagaag agaaaggtgg aggccagcta
     5461 agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5521 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5581 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5641 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag
     5701 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     5761 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     5821 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     5881 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     5941 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     6001 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     6061 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     6121 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     6181 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     6241 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     6301 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     6361 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6421 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     6481 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     6541 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     6601 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     6661 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6721 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6781 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6841 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6901 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6961 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     7021 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     7081 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     7141 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     7201 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     7261 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     7321 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     7381 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     7441 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag
     7501 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     7561 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     7621 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     7681 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7741 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7801 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7861 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7921 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     7981 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     8041 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     8101 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     8161 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     8221 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     8281 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     8341 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     8401 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
