LOCUS       sequence-202221-       12083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Confers constitutive expression of SpCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12083)
  AUTHORS   Shen MW, Arbab M, Hsu JY, Worstell D, Culbertson SJ, Krabbe O, Cassa
            CA, Liu DR, Gifford DK, Sherwood RI
  TITLE     Predictable and precise template-free CRISPR editing of pathogenic 
            variants.
  JOURNAL   Nature. 2018 Nov;563(7733):646-651. doi: 10.1038/s41586-018-0686-x. 
            Epub 2018 Nov 7.
  PUBMED    30405244
REFERENCE   2  (bases 1 to 12083)
  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"; doi: 
            "10.1038/s41586-018-0686-x"; journalName: "Nature"; date: "2018-11";
            volume: "563"; issue: "7733"; pages: "646-651"
COMMENT     Sequence Label: p2T-CAG-SpCas9-BlastR
FEATURES             Location/Qualifiers
     source          1..12083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(34..462)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     356..375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     589..611
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     603..620
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     604..620
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(679..690,691..853)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      878..887
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(893..926)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        934..1313
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1315..1592
                     /label=chicken beta-actin promoter
     intron          1594..2611
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2535..2557
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2619..2638
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2743..2759
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2744..2760
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2775..3060
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        3062..3339
                     /label=chicken beta-actin promoter
     intron          3340..3568
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      3580..3589
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3589..3654
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3661..3681
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3706..7806
                     /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             3706..7806
                     /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             7807..7854
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(7882..7899)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7888..8095
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    8119..8240
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    8508..8629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(8787..9185)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(9204..9251)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(9299..9628)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9313..9448
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9367..9386)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     9613..9633
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(9643..9830,9831..9842)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(9899..9919)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9899..9917)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9938..9954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9938..9954)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9951..9973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9962..9978
                     /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(9986..9992,9993..10010,10011..10016))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10031..10052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10169..10186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10340..10928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10420..10439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11099..11890,11891..11959))
                     /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     11722..11741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11960..12064)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
      421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 ccggcatatg gttcttgaca gaggtgtaaa aagtactcaa aaattttact caagtgaaag
      721 tacaagtact tagggaaaat tttactcaat taaaagtaaa agtatctggc tagaatctta
      781 cttgagtaaa agtaaaaaag tactccatta aaattgtact tgagtattaa ggaagtaaaa
      841 gtaaaagcaa gaaagatcga tctcgaagga tctggaggcc accatggtgt cgataacttc
      901 gtatagcata cattatacga agttatcgtg ctcgacattg attattgact agttattaat
      961 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     1021 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     1081 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     1141 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     1201 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     1261 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
     1321 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
     1381 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg
     1441 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg
     1501 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
     1561 ggccctataa aaagcgaagc gcgcggcggg cggggagtcg ctgcgacgct gccttcgccc
     1621 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
     1681 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta
     1741 atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc
     1801 tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc
     1861 gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc
     1921 gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg
     1981 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     2041 gggcgcgtcg gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg
     2101 cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg
     2161 ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc
     2221 gggggagggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     2281 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     2341 tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg
     2401 gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg
     2461 tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg
     2521 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
     2581 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct
     2641 gtctcatcat tttggcaaag aattcctcga gcggccgcca gtgtgatgga tatctgcaga
     2701 attcgaattc cagcacactg gcggccgtta ctcgaagtta tctcgaggtc gacggtatcg
     2761 ataagcttgg tacccgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     2821 gacccccgcc cattgacgtc aatagtaacg ccaataggga ctttccattg acgtcaatgg
     2881 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     2941 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattgtgc ccagtacatg
     3001 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     3061 gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca
     3121 attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg
     3181 gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt
     3241 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
     3301 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gacgctgcct
     3361 tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc
     3421 gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag
     3481 caagaggtaa gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga
     3541 gcacctgcct gaaatcactt tttttcaggt tggaccggtg ccaccatgga ctataaggac
     3601 cacgacggag actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc
     3661 ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc
     3721 atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag
     3781 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac
     3841 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga
     3901 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc
     3961 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg
     4021 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg
     4081 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc
     4141 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc
     4201 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
     4261 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg
     4321 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc
     4381 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg
     4441 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg
     4501 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
     4561 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg
     4621 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac
     4681 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag
     4741 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga
     4801 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc
     4861 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac
     4921 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag
     4981 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc
     5041 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc
     5101 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct
     5161 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag
     5221 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa
     5281 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag
     5341 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag
     5401 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
     5461 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
     5521 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
     5581 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
     5641 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg
     5701 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
     5761 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag
     5821 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
     5881 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
     5941 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
     6001 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
     6061 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
     6121 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
     6181 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt
     6241 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag
     6301 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg
     6361 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc
     6421 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag
     6481 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat
     6541 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
     6601 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
     6661 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
     6721 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
     6781 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc
     6841 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac
     6901 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg
     6961 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc
     7021 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg
     7081 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg
     7141 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc
     7201 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc
     7261 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg
     7321 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg
     7381 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag
     7441 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg
     7501 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat
     7561 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc
     7621 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac
     7681 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc
     7741 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga
     7801 ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa
     7861 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     7921 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     7981 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     8041 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc
     8101 cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     8161 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     8221 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     8281 gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaaaattgc
     8341 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac
     8401 ctctacaaat gtggtaaaat cgcgataagg atccgggctg gcgtaatagc gaagaggccc
     8461 gcaccgatcg cccttcccaa cagttgcgca gcctgaaaca gacatgataa gatacattga
     8521 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     8581 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga
     8641 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     8701 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt agcacgtgtc
     8761 agtcctgctc ctcggccacg aagtgcttag ccctcccaca cataaccaga gggcagcaat
     8821 tcacgaatcc caactgccgt cggctgtcca tcactgtcct tcactatggc tttgatccca
     8881 ggatgcagat cgagaagcac ctgtcggcac cgtccgcagg ggctcaagat gcccctgttc
     8941 tcatttccga tcgcgacgat acaagtcagg ttgccagctg ccgcagcagc agcagtgccc
     9001 agcaccacga gttctgcaca aggtccccca gtaaaatgat atacattgac accagtgaag
     9061 atgcggccgt cgctagagag agctgcgctg gcgacgctgt agtcttcaga gatggggatg
     9121 ctgttgattg tagccgttgc tctttcaatg agggtggatt cttcttgaga caaaggcttg
     9181 gccatggttt agttcctcac cttgtcgtat tatactatgc cgatatacta tgccgatgat
     9241 taattgtcaa cacgtgctga tcagatccga aaatggatat acaagctccc gggagctttt
     9301 tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggcag
     9361 aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
     9421 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
     9481 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     9541 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     9601 gggactttcc acaccctaac tgacacacat tccacagaat tcaagtgatc tccaaaaaat
     9661 aagtactttt tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa
     9721 aatgtaatta agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat
     9781 ccccaaaaat aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc
     9841 tggttcttga ccccctacct tcagcaagcc cagcagatcc gagctccagc ttttgttccc
     9901 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     9961 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
    10021 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
    10081 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
    10141 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
    10201 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
    10261 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
    10321 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
    10381 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
    10441 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
    10501 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
    10561 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
    10621 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
    10681 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
    10741 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
    10801 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
    10861 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
    10921 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
    10981 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
    11041 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
    11101 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
    11161 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
    11221 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
    11281 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
    11341 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
    11401 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
    11461 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
    11521 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
    11581 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
    11641 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
    11701 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
    11761 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
    11821 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
    11881 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
    11941 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
    12001 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
    12061 cgcgcacatt tccccgaaaa gtg
//
