LOCUS       sequence-105859-        9475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FokI-dCas9 and gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9475)
  AUTHORS   Nakagawa Y, Sakuma T, Sakamoto T, Ohmuraya M, Nakagata N, Yamamoto T
  TITLE     Production of knockout mice by DNA microinjection of various 
            CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes.
  JOURNAL   BMC Biotechnol. 2015 May 22;15(1):33.
  PUBMED    25997509
REFERENCE   2  (bases 1 to 9475)
  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: "BMC 
            Biotechnol."; date: "2015-05-22"; volume: "15(1)"; pages: "33"
COMMENT     Sequence Label: pX330A_FokI-1x4
FEATURES             Location/Qualifiers
     source          1..9475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        295..572
                     /label=chicken beta-actin promoter
     intron          573..801
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             822..842
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             849..1436
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             join(1452..1475,1476..1478,1479..3965,3966..3968,
                     3969..5552)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1452..5552
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5553..5600
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5628..5645)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5634..5841
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5850..5990
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5850..5979
                     /label=AAV2 ITR
     rep_origin      6065..6520
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6152..6171)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6362..6383
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6537..6556)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6656..6678
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6716..6734)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6802..6906
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6907..6975,6976..7767)
                     /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(7125..7144)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7938..8526
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8427..8446
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8588..8836
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8588..8608
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8759..8778
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8855..8930
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(9016..9321)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     9171..9193
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9185..9202
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     9186..9202
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9212..9231
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9212..9230
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9278..9298)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9278..9296)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9317..9333)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9317..9333)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9330..9352)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9341..9357
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(9365..9371,9372..9389,9390..9395))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9410..9431
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
       61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
      241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg
      421 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg
      481 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
      541 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc
      601 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
      661 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg
      721 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg
      781 cctgaaatca ctttttttca ggttggaccg gtgccaccat gcctaagaag aagcggaagg
      841 tgagctccca actcgtgaag agtgaacttg aggagaaaaa gtcggagctg cggcacaaat
      901 tgaaatacgt accgcatgaa tacatcgaac ttatcgaaat tgctaggaac tcgactcaag
      961 acagaatcct tgagatgaag gtaatggagt tctttatgaa ggtttatgga taccgaggga
     1021 agcatctcgg tggatcacga aaacccgacg gagcaatcta tacggtgggg agcccgattg
     1081 attacggagt gatcgtcgac acgaaagcct acagcggtgg gtacaatctt cccatcgggc
     1141 aggcagatga gatgcaacgt tatgtcgaag aaaatcagac caggaacaaa cacatcaatc
     1201 caaatgagtg gtggaaagtg tatccttcat cagtgaccga gtttaagttt ttgtttgtct
     1261 ctgggcattt caaaggcaac tataaggccc agctcacacg gttgaatcac attacgaact
     1321 gcaatggtgc ggttttgtcc gtagaggaac tgctcattgg tggagaaatg atcaaagcgg
     1381 gaactctgac actggaagaa gtcagacgca agtttaacaa tggcgagatc aatttcggtg
     1441 gcggtggatc cgacaagaag tacagcatcg gcctggccat cggcaccaac tctgtgggct
     1501 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
     1561 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa
     1621 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
     1681 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
     1741 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
     1801 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
     1861 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
     1921 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     1981 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     2041 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     2101 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     2161 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     2221 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     2281 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     2341 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     2401 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     2461 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     2521 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     2581 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     2641 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     2701 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     2761 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     2821 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     2881 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact
     2941 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     3001 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     3061 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga
     3121 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     3181 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     3241 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     3301 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     3361 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     3421 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
     3481 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
     3541 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     3601 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     3661 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     3721 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     3781 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     3841 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     3901 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     3961 tggacgccat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
     4021 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     4081 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
     4141 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     4201 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     4261 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
     4321 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     4381 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
     4441 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     4501 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     4561 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     4621 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
     4681 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
     4741 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
     4801 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     4861 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     4921 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     4981 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     5041 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     5101 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     5161 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     5221 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     5281 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     5341 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     5401 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     5461 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     5521 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg
     5581 gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc
     5641 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     5701 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     5761 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     5821 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     5881 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     5941 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     6001 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     6061 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     6121 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     6181 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     6241 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     6301 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     6361 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat
     6421 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     6481 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     6541 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     6601 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     6661 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     6721 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     6781 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6841 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     6901 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6961 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     7021 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     7081 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     7141 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     7201 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     7261 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     7321 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     7381 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     7441 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     7501 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     7561 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     7621 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     7681 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     7741 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     7801 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     7861 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     7921 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     7981 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     8041 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     8101 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     8161 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     8221 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     8281 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     8341 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     8401 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     8461 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     8521 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     8581 cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     8641 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     8701 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     8761 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     8821 gaaaggacga aacaccgggt cttcgagaag acctgtttta gagctagaaa tagcaagtta
     8881 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt
     8941 tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt gcgccaattc
     9001 tgcagacaaa tggctctaga gaccggcgcc gctacagggc gcgtcccatt cgccattcag
     9061 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     9121 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     9181 acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg
     9241 taccgggccc cccctcgagg tcctccagct tttgttccct ttagtgaggg ttaattgcgc
     9301 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     9361 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     9421 aactcacatt aattgcgttg cgctcactgc ccgctttcca ccggtggtct cttag
//
