LOCUS       sequence-340295-        9136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A knockout vector for the dog Tgfa..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9136)
  AUTHORS   Lin S, Hirayama D, Maryu G, Matsuda K, Hino N, Deguchi E, Aoki K, 
            Iwamoto R, Terai K, Matsuda M
  TITLE     Redundant roles of EGFR ligands in the ERK activation waves during 
            collective cell migration.
  JOURNAL   Life Sci Alliance. 2021 Oct 19;5(1). pii: 5/1/e202101206. doi: 
            10.26508/lsa.202101206. Print 2022 Jan.
  PUBMED    34667080
REFERENCE   2  (bases 1 to 9136)
  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: "Life Sci 
            Alliance."; date: "2021-10-19"; volume: "5(1). pii: 5/1/e202101206. 
            doi"; pages: " 10.26508/lsa.202101206. Print 2022 Jan"
COMMENT     Sequence Label: pX459-dTgfa
FEATURES             Location/Qualifiers
     source          1..9136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          121..348
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      360..369
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             369..434
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             441..461
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             486..4586
                     /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             486..4586
                     /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             4587..4634
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4650..4703
                     /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             4704..5303
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4704..4723)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(5331..5348)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5337..5544
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5553..5693
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5553..5682
                     /label=AAV2 ITR
     rep_origin      5768..6223
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5855..5874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6065..6086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6240..6259)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6359..6381
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6419..6437)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6505..6609
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6610..6678,6679..7470)
                     /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(6828..6847)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7641..8229
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8130..8149
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8291..8539
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8291..8311
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8462..8481
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8561..8636
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8733..9018
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
       61 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      121 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      181 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      241 ccgggctgta attagctgag caagaggtaa gggtttaagg gatggttggt tggtggggta
      301 ttaatgttta attacctgga gcacctgcct gaaatcactt tttttcaggt tggaccggtg
      361 ccaccatgga ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag
      421 acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag
      481 cagccgacaa gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg
      541 tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc
      601 ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg
      661 aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct
      721 gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca
      781 gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg
      841 gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa
      901 agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc
      961 acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg
     1021 acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc
     1081 ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca
     1141 gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa
     1201 acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg
     1261 aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg
     1321 cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca
     1381 tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct
     1441 ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc
     1501 ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg
     1561 ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc
     1621 tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc
     1681 ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc
     1741 acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga
     1801 tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca
     1861 gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg
     1921 aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata
     1981 agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg
     2041 tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc
     2101 tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga
     2161 ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa
     2221 tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa
     2281 ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg
     2341 tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg
     2401 cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg
     2461 gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc
     2521 tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg
     2581 acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc
     2641 tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga
     2701 cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg
     2761 tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga
     2821 gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc
     2881 ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc
     2941 gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc
     3001 atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa
     3061 gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga
     3121 agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc
     3181 tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac
     3241 agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga
     3301 acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt
     3361 ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca
     3421 actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa
     3481 agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga
     3541 agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca
     3601 gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc
     3661 ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt
     3721 ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg
     3781 tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga
     3841 tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg
     3901 cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg
     3961 tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg
     4021 actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta
     4081 agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac
     4141 tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca
     4201 gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg
     4261 aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag
     4321 tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata
     4381 agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag
     4441 cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca
     4501 aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga
     4561 tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg
     4621 caaaaaagaa aaaggaattc ggcagtggag agggcagagg aagtctgcta acatgcggtg
     4681 acgtcgagga gaatcctggc ccaatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     4741 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     4801 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     4861 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     4921 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     4981 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     5041 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     5101 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     5161 gcgccggggt gcccgccttc ctggagacct ccgcgcccca caacctcccc ttctacgagc
     5221 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     5281 tgacccgcaa gcccggtgcc tgagaattct aactagagct cgctgatcag cctcgactgt
     5341 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     5401 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     5461 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     5521 agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg agttggccac
     5581 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     5641 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     5701 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     5761 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     5821 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     5881 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5941 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     6001 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     6061 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     6121 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     6181 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     6241 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     6301 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     6361 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     6421 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     6481 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     6541 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     6601 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     6661 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     6721 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     6781 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     6841 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     6901 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     6961 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     7021 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     7081 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     7141 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     7201 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     7261 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     7321 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     7381 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     7441 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     7501 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     7561 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     7621 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     7681 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     7741 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     7801 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     7861 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     7921 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     7981 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     8041 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     8101 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     8161 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     8221 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     8281 gctcacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     8341 tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     8401 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     8461 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     8521 gtggaaagga cgaaacaccg gtcccacttc aacgactgcc gttttagagc tagaaatagc
     8581 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     8641 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc
     8701 caattctgca gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg
     8761 cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact
     8821 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     8881 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     8941 cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     9001 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
     9061 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
     9121 ggggcggggg gggggg
//
