LOCUS       sequence-447852-        9242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pX459 plasmid encoding the sgRNA protospacer sequence 
            "5'-GCATAATGTCACTACAGATC-3' to target the LIG4 gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9242)
  AUTHORS   Cortazar MA, Jagannathan S
  TITLE     SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through 
            Selectable Homology-Directed DNA Repair.
  JOURNAL   Methods Mol Biol. 2025;2863:397-417. doi: 
            10.1007/978-1-0716-4176-7_23.
  PUBMED    39535722
REFERENCE   2  (bases 1 to 9242)
  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: "Methods Mol
            Biol."; date: "2025"; volume: "2863:397-417. doi"; pages: " 
            10.1007/978-1-0716-4176-7_23"
COMMENT     Sequence Label: pX459_LIG4_Exon3
FEATURES             Location/Qualifiers
     source          1..9242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(3082..3102)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3109..3174)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      3174..3183
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     enhancer        3768..4053
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     misc_RNA        complement(4150..4225)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4246..4494)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4304..4323)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4474..4494)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     rep_origin      complement(4556..5144)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4636..4655)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5315..6106,6107..6175))
                     /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     5938..5957
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6176..6280)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6348..6366
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6404..6426)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6526..6545
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6562..7017)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6699..6720)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6911..6930
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   7092..7232
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7103..7232
                     /label=AAV2 ITR
     polyA_signal    7241..7448
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7437..7454
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(7482..8081)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7565..7585)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     8062..8081
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(8082..8135)
                     /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             complement(8151..8198)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(join(8199..9242,1..3057))
                     /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             complement(join(8199..9242,1..3057))
                     /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"
ORIGIN
        1 cttccgcacg tcgtacacct tgtagtcgcc gtacacgaac tcgctttcca gcttagggta
       61 ctttttgatc agggcggttc ccacgacggc gttcaggtag gcgtcgtggg cgtggtggta
      121 gttgttgatc tcgcgcactt tgtaaaactg gaaatccttc cggaaatcgg acaccagctt
      181 ggacttcagg gtgatcactt tcacttcccg gatcagcttg tcattctcgt cgtacttagt
      241 gttcatccgg gagtccagga tctgtgccac gtgctttgtg atctgccggg tttccaccag
      301 ctgtctcttg atgaagccgg ccttatccag ttcgctcagg ccgcctctct cggccttggt
      361 cagattgtcg aactttctct gggtaatcag cttggcgttc agcagctgcc gccagtagtt
      421 cttcatcttc ttcacgacct cttcggaggg cacgttgtcg ctcttgcccc ggttcttgtc
      481 gcttctggtc agcaccttgt tgtcgatgga gtcgtccttc agaaagctct gaggcacgat
      541 atggtccaca tcgtagtcgg acagccggtt gatgtccagt tcctggtcca cgtacatatc
      601 ccgcccattc tgcaggtagt acaggtacag cttctcgttc tgcagctggg tgttttccac
      661 ggggtgttct ttcaggatct ggctgcccag ctctttgatg ccctcttcga tccgcttcat
      721 tctctcgcgg ctgttcttct gtcccttctg ggtggtctgg ttctctctgg ccatttcgat
      781 cacgatgttc tcgggcttgt gccggcccat cactttcacg agctcgtcca ccaccttcac
      841 tgtctgcagg atgcccttct taatggcggg gctgccggcc agattggcaa tgtgctcgtg
      901 caggctatcg ccctggccgg acacctgggc tttctggatg tcctctttaa aggtcaggct
      961 gtcgtcgtgg atcagctgca tgaagtttct gttggcgaag ccgtcggact tcaggaaatc
     1021 caggattgtc ttgccggact gcttgtcccg gatgccgttg atcagcttcc ggctcagcct
     1081 gccccagccg gtgtatctcc gccgcttcag ctgcttcatc actttgtcgt cgaacaggtg
     1141 ggcataggtt ttcagccgtt cctcgatcat ctctctgtcc tcaaacagtg tcagggtcag
     1201 cacgatatct tccagaatgt cctcgttttc ctcattgtcc aggaagtcct tgtccttgat
     1261 aattttcagc agatcgtggt atgtgcccag ggaggcgttg aaccgatctt ccacgccgga
     1321 gatttccacg gagtcgaagc actcgatttt cttgaagtag tcctctttca gctgcttcac
     1381 ggtcactttc cggttggtct tgaacagcag gtccacgatg gcctttttct gctcgccgct
     1441 caggaaggcg ggctttctca ttccctcggt cacgtatttc actttggtca gctcgttata
     1501 cacggtgaag tactcgtaca gcaggctgtg cttgggcagc accttctcgt tgggcaggtt
     1561 cttatcgaag ttggtcatcc gctcgatgaa gctctgggcg gaagcgccct tgtccaccac
     1621 ttcctcgaag ttccaggggg tgatggtttc ctcgctcttt ctggtcatcc aggcgaatct
     1681 gctgtttccc ctggccagag ggcccacgta gtaggggatg cggaaggtca ggatcttctc
     1741 gatcttttcc cggttgtcct tcaggaatgg gtaaaaatct tcctgccgcc gcagaatggc
     1801 gtgcagctct cccaggtgga tctggtgggg gatgctgccg ttgtcgaagg tccgctgctt
     1861 ccgcagcagg tcctctctgt tcagcttcac gagcagttcc tcggtgccgt ccatcttttc
     1921 caggatgggc ttgatgaact tgtagaactc ttcctggctg gctccgccgt caatgtagcc
     1981 ggcgtagccg ttcttgctct ggtcgaagaa aatctctttg tacttctcag gcagctgctg
     2041 ccgcacgaga gctttcagca gggtcaggtc ctggtggtgc tcgtcgtatc tcttgatcat
     2101 agaggcgctc aggggggcct tggtgatctc ggtgttcact ctcaggatgt cgctcagcag
     2161 gatggcgtcg gacaggttct tggcggccag aaacaggtcg gcgtactggt cgccgatctg
     2221 ggccagcagg ttgtccaggt cgtcgtcgta ggtgtccttg ctcagctgca gtttggcatc
     2281 ctcggccagg tcgaagttgc tcttgaagtt gggggtcagg cccaggctca gggcaatcag
     2341 gtttccgaac aggccattct tcttctcgcc gggcagctgg gcgatcagat tttccagccg
     2401 tctgctcttg ctcagtctgg cagacaggat ggccttggcg tccacgccgc tggcgttgat
     2461 ggggttttcc tcgaacagct ggttgtaggt ctgcaccagc tggatgaaca gcttgtccac
     2521 gtcgctgttg tcggggttca ggtcgccctc gatcaggaag tggccccgga acttgatcat
     2581 gtgggccagg gccagataga tcagccgcag gtcggccttg tcggtgctgt ccaccagttt
     2641 ctttctcagg tggtagatgg tggggtactt ctcgtggtag gccacctcgt ccacgatgtt
     2701 gccgaagatg gggtgccgct cgtgcttctt atcctcttcc accaggaagg actcttccag
     2761 tctgtggaag aagctgtcgt ccaccttggc catctcgttg ctgaagatct cttgcagata
     2821 gcagatccgg ttcttccgtc tggtgtatct tcttctggcg gttctcttca gccgggtggc
     2881 ctcggctgtt tcgccgctgt cgaacagcag ggctccgatc aggttcttct tgatgctgtg
     2941 ccggtcggtg ttgcccagca ccttgaattt cttgctgggc accttgtact cgtcggtgat
     3001 cacggcccag cccacagagt tggtgccgat gtccaggccg atgctgtact tcttgtcggc
     3061 tgctgggact ccgtggatac cgaccttccg cttcttcttt ggggccatct tatcgtcatc
     3121 gtctttgtaa tcaatatcat gatccttgta gtctccgtcg tggtccttat agtccatggt
     3181 ggcaccggtc caacctgaaa aaaagtgatt tcaggcaggt gctccaggta attaaacatt
     3241 aataccccac caaccaacca tcccttaaac ccttacctct tgctcagcta attacagccc
     3301 ggaggagaag ggccgtcccg cccgctcacc tgtgggagta acgcggtcag tcagagccgg
     3361 ggcgggcggc gcgaggcggc ggcggagcgg ggcacggggc gaaggcagcg cgcagcgacc
     3421 cccgcccgcc gcgcgcttcg ctttttatag ggccgccgcc gccgccgcct cgccctaaaa
     3481 ggaaactttc ggagcgcgcc gccctgattg gctgccgccg cacctctccg cccccccccc
     3541 cccccccccc cccccccgcc cccccccccc cccccccccc ccccccgccc cccccccccc
     3601 cccccccccc cccccccccc ccccccccgc cccgcccggg gggccccccc cccccccccc
     3661 gcccccatcg ctgcacaaaa taattaaaaa ataaataaat acaaaattgg gggtggggag
     3721 gggggggaga tggggagagt gaagcagaac gtggggctca cctcgaccat ggtaatagcg
     3781 atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca
     3841 caatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga
     3901 tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa
     3961 tggaaagtcc ctattggcgt tactattgac gtcaatgggc gggggtcgtt gggcggtcag
     4021 ccaggcgggc catttaccgt aagttatgta acgggtacct ctagagccat ttgtctgcag
     4081 aattggcgca cgcgctaaaa acggactagc cttattttaa cttgctattt ctagctctaa
     4141 aacaaaaaag caccgactcg gtgccacttt ttcaagttga taacggacta gccttatttt
     4201 aacttgctat ttctagctct aaaacgatct gtagtgacat tatgcggtgt ttcgtccttt
     4261 ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa gcatatgata
     4321 gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt actttctacg
     4381 tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattccaa ttatctctct
     4441 aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc cctcacatgt
     4501 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4561 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4621 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4681 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4741 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4801 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4861 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4921 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4981 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5041 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5101 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5161 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5221 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5281 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5341 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5401 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcggcttc
     5461 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5521 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5581 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5641 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5701 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5761 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5821 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5881 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5941 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6001 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6061 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6121 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6181 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6241 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6301 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     6361 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     6421 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     6481 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     6541 ttgtactgag agtgcaccat aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa
     6601 atttttgtta aatcagctca ttttttaacc aatagaccga aatcggcaaa atcccttata
     6661 aatcaaaaga atagcccgag atagagttga gtgttgttcc agtttggaac aagagtccac
     6721 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     6781 cactacgtga accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     6841 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     6901 cgagaaagga agggaagaaa gcgaaaggag cgggcgctaa ggcgctggca agtgtagcgg
     6961 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtact
     7021 atggttgctt tgacgtatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     7081 catcaggcgc ccctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa
     7141 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
     7201 gggagtggcc aactccatca ctaggggttc ctgcggccgc tccccagcat gcctgctatt
     7261 ctcttcccaa tcctccccct tgctgtcctg ccccacccca ccccccagaa tagaatgaca
     7321 cctactcaga caatgcgatg caatttcctc attttattag gaaaggacag tgggagtggc
     7381 accttccagg gtcaaggaag gcacggggga ggggcaaaca acagatggct ggcaactaga
     7441 aggcacagtc gaggctgatc agcgagctct agttagaatt ctcaggcacc gggcttgcgg
     7501 gtcatgcacc aggtgcgcgg tccttcgggc acctcgacgt cggcggtgac ggtgaagccg
     7561 agccgctcgt agaaggggag gttgcggggc gcggaggtct ccaggaaggc gggcaccccg
     7621 gcgcgctcgg ccgcctccac tccggggagc acgacggcgc tgcccagacc cttgccctgg
     7681 tggtcgggcg agactccgac ggtggccagg aaccacgcgg gctccttggg ccggtgcggc
     7741 gccaggaggc cttccatctg ttgctgcgcg gccagccggg aaccgctcaa ctcggccatg
     7801 cgcgggccga tctcggcgaa caccgccccc gcttcgacgc tctccggcgt ggtccagacc
     7861 gccaccgcgg cgccgtcgtc cgcgacccac accttgccga tgtcgagccc gacgcgcgtg
     7921 aggaagagtt cttgcagctc ggtgacccgc tcgatgtggc ggtccggatc gacggtgtgg
     7981 cgcgtggcgg ggtagtcggc gaacgcggcg gcgagggtgc gtacggccct ggggacgtcg
     8041 tcgcgggtgg cgaggcgcac cgtgggcttg tactcggtca ttgggccagg attctcctcg
     8101 acgtcaccgc atgttagcag acttcctctg ccctctccac tgccgaattc ctttttcttt
     8161 tttgcctggc cggccttttt cgtggccgcc ggccttttgt cgcctcccag ctgagacagg
     8221 tcgatccgtg tctcgtacag gccggtgatg ctctggtgga tcagggtggc gtccagcacc
     8281 tctttggtgc tggtgtacct cttccggtcg atggtggtgt caaagtactt gaaggcggca
     8341 ggggctccca gattggtcag ggtaaacagg tggatgatat tctcggcctg ctctctgatg
     8401 ggcttatccc ggtgcttgtt gtaggcggac agcactttgt ccagattagc gtcggccagg
     8461 atcactctct tggagaactc gctgatctgc tcgatgatct cgtccaggta gtgcttgtgc
     8521 tgttccacaa acagctgttt ctgctcatta tcctcggggg agcccttcag cttctcatag
     8581 tggctggcca ggtacaggaa gttcacatat ttggagggca gggccagttc gtttcccttc
     8641 tgcagttcgc cggcagaggc cagcattctc ttccggccgt tttccagctc gaacagggag
     8701 tacttaggca gcttgatgat caggtccttt ttcacttctt tgtagccctt ggcttccaga
     8761 aagtcgatgg gattcttctc gaagctgctt ctttccatga tggtgatccc cagcagctct
     8821 ttcacactct tcagtttctt ggacttgccc ttttccactt tggccaccac cagcacagaa
     8881 taggccacgg tggggctgtc gaagccgccg tacttcttag ggtcccagtc cttctttctg
     8941 gcgatcagct tatcgctgtt cctcttgggc aggatagact ctttgctgaa gccgcctgtc
     9001 tgcacctcgg tctttttcac gatattcact tggggcatgc tcagcacttt ccgcacggtg
     9061 gcaaaatccc ggcccttatc ccacacgatc tccccggttt cgccgtttgt ctcgatcaga
     9121 ggccgcttcc ggatctcgcc gttggccagg gtaatctcgg tcttgaaaaa gttcatgatg
     9181 ttgctgtaga agaagtactt ggcggtagcc ttgccgattt cctgctcgct cttggcgatc
     9241 at
//
