LOCUS       sequence-259492-        9285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for sgRNAs cloned into the BsaI sites and for 
            expression of Cas9 linked to mCherry via a T2A peptide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9285)
  AUTHORS   Reuven N, Adler J, Broennimann K, Myers N, Shaul Y
  TITLE     Recruitment of DNA Repair MRN Complex by Intrinsically Disordered 
            Protein Domain Fused to Cas9 Improves Efficiency of CRISPR-Mediated 
            Genome Editing.
  JOURNAL   Biomolecules. 2019 Oct 8;9(10). pii: biom9100584. doi: 
            10.3390/biom9100584.
  PUBMED    31597252
REFERENCE   2  (bases 1 to 9285)
  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: 
            "Biomolecules."; date: "2019-10-8"; volume: "9(10). pii: 
            biom9100584. doi"; pages: " 10.3390/biom9100584"
COMMENT     Sequence Label: pU6-(BsaI)_CBh-Cas9-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..9285
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7..27
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     178..197
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        276..351
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        448..733
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        735..1012
                     /label=chicken beta-actin promoter
     intron          1013..1241
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1253..1262
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1262..1327
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1334..1354
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1379..5479
                     /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="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             1379..5479
                     /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="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             5480..5527
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5531..5584
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5591..6301
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5736..5754)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(6326..6343)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6332..6539
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6548..6688
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6548..6677
                     /label=AAV2 ITR
     rep_origin      6763..7218
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6850..6869)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7060..7081
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7235..7254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7354..7376
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7414..7432)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7500..7604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7605..7673,7674..8465)
                     /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(7823..7842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8636..9224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9125..9144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccccacgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccggaga cctgtacagg tctctgtttt 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 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
      841 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
      901 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
      961 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
     1021 tgcgacgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     1081 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     1141 gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt
     1201 ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat
     1261 ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga
     1321 cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga
     1381 caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac
     1441 cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag
     1501 catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac
     1561 ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct
     1621 gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga
     1681 agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat
     1741 cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact
     1801 ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat
     1861 caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga
     1921 caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa
     1981 cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct
     2041 ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat
     2101 tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc
     2161 caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat
     2221 cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct
     2281 gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat
     2341 caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca
     2401 gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta
     2461 cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa
     2521 gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca
     2581 gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat
     2641 tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa
     2701 gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt
     2761 cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt
     2821 ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct
     2881 gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa
     2941 cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg
     3001 cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa
     3061 gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg
     3121 cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa
     3181 ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac
     3241 cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct
     3301 gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct
     3361 gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt
     3421 cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct
     3481 gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga
     3541 gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa
     3601 ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga
     3661 aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa
     3721 gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga
     3781 aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat
     3841 gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt
     3901 gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa
     3961 gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta
     4021 ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa
     4081 ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt
     4141 ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa
     4201 gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct
     4261 ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca
     4321 ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc
     4381 taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat
     4441 cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat
     4501 catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct
     4561 gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac
     4621 cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac
     4681 aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag
     4741 aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc
     4801 tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga
     4861 gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct
     4921 ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc
     4981 cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa
     5041 gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta
     5101 tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca
     5161 caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct
     5221 ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat
     5281 cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc
     5341 cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt
     5401 gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct
     5461 gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa
     5521 gaaaaagctt gagggcagag gaagtctgct aacatgcggt gacgtggagg agaatcccgg
     5581 ccctgctagc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat
     5641 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     5701 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     5761 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
     5821 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     5881 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     5941 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctcaga
     6001 cggccccgta atgcagaaga aaaccatggg ctgggaggcc tcctccgagc ggatgtaccc
     6061 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     6121 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     6181 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
     6241 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta
     6301 agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     6361 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     6421 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     6481 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag
     6541 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     6601 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     6661 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     6721 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     6781 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     6841 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     6901 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     6961 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     7021 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     7081 gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     7141 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     7201 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     7261 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     7321 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     7381 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     7441 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     7501 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     7561 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     7621 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     7681 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     7741 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     7801 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     7861 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     7921 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     7981 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     8041 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     8101 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     8161 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     8221 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     8281 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag
     8341 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     8401 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     8461 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     8521 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     8581 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     8641 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     8701 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     8761 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     8821 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     8881 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     8941 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     9001 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     9061 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     9121 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     9181 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     9241 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
