LOCUS       sequence-233471-        8447 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 8447)
  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 8447)
  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..8447
                     /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"
     promoter        740..1017
                     /label=chicken beta-actin promoter
     intron          1018..1246
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1267..1293
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1297..1317
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1327..1350,1351..1353,1354..5427)
                     /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             1327..5427
                     /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             5431..5451
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5488..5505)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5494..5701
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5710..5850
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5710..5839
                     /label=AAV2 ITR
     rep_origin      5925..6380
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6012..6031)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6222..6243
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6397..6416)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6516..6538
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6576..6594)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6662..6766
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6767..6835,6836..7627)
                     /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(6985..7004)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7798..8386
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8287..8306
                     /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 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc
      901 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      961 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
     1021 gtcgctgcga cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
     1081 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
     1141 gggctgtaat tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt
     1201 aatgtttaat tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc
     1261 accatgtacc catacgatgt tccagattac gcttcgccga agaaaaagcg caaggtcgaa
     1321 gcgtccgaca agaagtacag catcggcctg gccatcggca ccaactctgt gggctgggcc
     1381 gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac
     1441 cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc
     1501 gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc
     1561 tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac
     1621 agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc
     1681 ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga
     1741 aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc
     1801 cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc
     1861 gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac
     1921 cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc
     1981 agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcggc
     2041 aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc
     2101 gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg
     2161 gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc
     2221 atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc
     2281 tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg
     2341 cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac
     2401 gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc
     2461 ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg
     2521 cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg
     2581 cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag
     2641 atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac
     2701 agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag
     2761 gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat
     2821 aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc
     2881 gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc
     2941 ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg
     3001 accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa
     3061 atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa
     3121 attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc
     3181 gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat
     3241 gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg
     3301 ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc
     3361 ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac
     3421 gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc
     3481 ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag
     3541 acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc
     3601 gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag
     3661 agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac
     3721 cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg
     3781 cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac
     3841 catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga
     3901 agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg
     3961 aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat
     4021 ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga
     4081 cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg
     4141 aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag
     4201 tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac
     4261 aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa
     4321 aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg
     4381 aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac
     4441 agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag
     4501 cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat
     4561 tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag
     4621 gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg
     4681 atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg
     4741 gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt
     4801 gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc
     4861 gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct
     4921 aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa
     4981 ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc
     5041 agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg
     5101 gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga
     5161 gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat
     5221 aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga
     5281 gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc
     5341 aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg
     5401 atcgacctgt ctcagctggg aggcgacagc cccaagaaga agagaaaggt ggaggccagc
     5461 taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     5521 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     5581 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     5641 gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg
     5701 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     5761 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     5821 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     5881 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     5941 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     6001 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     6061 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     6121 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6181 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6241 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     6301 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     6361 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     6421 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     6481 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     6541 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     6601 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     6661 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     6721 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     6781 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     6841 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     6901 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     6961 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     7021 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     7081 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     7141 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     7201 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     7261 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     7321 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     7381 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     7441 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     7501 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     7561 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     7621 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     7681 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     7741 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     7801 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     7861 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     7921 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     7981 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     8041 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     8101 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     8161 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     8221 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     8281 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     8341 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     8401 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
