LOCUS       sequence-438782-       13266 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Sleeping beauty transposon with conditional expressed hCas9. A red
            flourescent gene linked to puromycin can be removed in the prescence
            of Flp recombinase allowing Cas9 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13266)
  AUTHORS   Berthelsen MF, Riedel M, Cai H, Skaarup SH, Alstrup AKO, 
            Dagnaes-Hansen F, Luo Y, Jensen UB, Hager H, Liu Y, Callesen H, 
            Vendelbo MH, Jakobsen JE, Thomsen MK
  TITLE     The CRISPR/Cas9 Minipig-A Transgenic Minipig to Produce Specific 
            Mutations in Designated Tissues.
  JOURNAL   Cancers (Basel). 2021 Jun 16;13(12). pii: cancers13123024. doi: 
            10.3390/cancers13123024.
  PUBMED    34208747
REFERENCE   2  (bases 1 to 13266)
  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: "Cancers 
            (Basel)."; date: "2021-06-16"; volume: "13(12). pii: 
            cancers13123024. doi"; pages: " 10.3390/cancers13123024"
COMMENT     Sequence Label: LIR_TRE_CAG_mKateII-Puro-STOP_mVenus-Cas9-PA-RIR
FEATURES             Location/Qualifiers
     source          1..13266
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    698..731
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    join(754..772,773..795,796..814,815..837,838..856,857..879,
                     880..898,899..921,922..940,941..963,964..982,983..1005,
                     1006..1024)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     enhancer        1100..1403
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1405..1685
                     /label=chicken beta-actin promoter
     primer_bind     2488..2510
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2575..2591
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2601..2620
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2601..2619
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2683..2702
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    2734..2781
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     regulatory      2782..2791
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2788..3483
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             3499..3558
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             3565..4164
                     /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(3565..3584)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4061..4081
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4211..4435
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4842..4889
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    4904..4935
                     /label=rox
                     /bound_moiety="Dre recombinase"
     regulatory      4946..4955
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4952..4954,4955..4957,4958..5668)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5258..5277)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5605..5626
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5678..5734
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5735..5800
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5807..5827
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5852..9952
                     /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             5852..9952
                     /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             9953..10000
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    10034..10241
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    10288..10512
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(10529..10562)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(11045..11061)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11045..11061)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11058..11080)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11069..11085
                     /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(11093..11099,11100..11117,11118..11123))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11138..11159
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11276..11293)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11447..12035)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11527..11546)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12206..12997,12998..13066))
                     /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     12829..12848
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13067..13171)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     13239..13257
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cagatctgat atctctagag
      421 aagtcgacgt ttaaagtacc ctacagttga agtcggaagt ttacatacac ttaagttgga
      481 gtcattaaaa ctcgtttttc aactactcca caaatttctt gttaacaaac aatagttttg
      541 gcaagtcagt taggacatct actttgtgca tgacacaagt catttttcca acaattgttt
      601 acagacagat tatttcactt ataattcact gtatcacaat tccagtgggt cagaagttta
      661 catacactaa gttgactgtg cctttaaaca gtcgcgaata acttcgtata atgtatgcta
      721 tacgaagtta tggcgcgccc tcgagtttac cactccctat cagtgataga gaaaagtgaa
      781 agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc
      841 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
      901 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
      961 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
     1021 gagaaaagtg aaagtcgact agttattaat agtaatcaat tacggggtca ttagttcata
     1081 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     1141 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     1201 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     1261 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     1321 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1381 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
     1441 tctccccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     1501 gcgatggggg cggggggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
     1561 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
     1621 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
     1681 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
     1741 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
     1801 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc
     1861 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg
     1921 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg
     1981 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg
     2041 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg
     2101 gtgtgtgcgt gggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
     2161 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
     2221 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
     2281 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
     2341 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
     2401 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
     2461 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     2521 gtaacggccg ccagtgtgct ggaattcgcc cttcaagctt ccagtcacga cgttgtaaaa
     2581 cgacggccag tgagcgcgcg taatacgact cactataggg cgaattggag ctcgcctctg
     2641 ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg
     2701 tgctgtctca tcattttggc aaagattaat taagaagttc ctattccgaa gttcctattc
     2761 tctagaaagt ataggaactt cgccaccatg gtgagcgagc tgattaagga gaacatgcac
     2821 atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc
     2881 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggcggtcga gggcggccct
     2941 ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa aaccttcatc
     3001 aaccacaccc agggcatccc cgacttcttt aagcagtcct tccccgaggg cttcacatgg
     3061 gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc
     3121 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc
     3181 cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagaccct gtaccccgct
     3241 gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg gggccacctg
     3301 atctgcaact tgaagaccac atacagatcc aagaaacccg ctaagaacct caagatgccc
     3361 ggcgtctact atgtggacag aagactggaa agaatcaagg aggccgacaa agagacctac
     3421 gtcgagcagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac
     3481 agacctaggg gaagcggaca gtgtactaat tatgctctct tgaaattggc tggagatgtt
     3541 gagagcaacc ctggacctac cggtatgacc gagtacaagc ccacggtgcg cctcgccacc
     3601 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     3661 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     3721 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     3781 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     3841 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     3901 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     3961 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     4021 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     4081 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     4141 atgacccgca agcccggtgc ctgaatcgat gactctagat cataatcagc ccgggggtga
     4201 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     4261 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     4321 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     4381 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggaacca
     4441 gctggttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4501 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4561 cttatcatgt ctggatcttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4621 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4681 tcatcaatgt atcttatcat gtctggatct tgtttattgc agcttataat ggttacaaat
     4741 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4801 gtttgtccaa actcatcaat gtatcttatc atgtctggat cgaagttcct attccgaagt
     4861 tcctattctc tagaaagtat aggaacttcc tcatgcgcat aactaacttt aaataatgcc
     4921 aattatttaa agttattgcg atcgcgccac catggtgagc aagggcgagg agctgttcac
     4981 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     5041 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagc tgatctgcac
     5101 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgggct acggcctgca
     5161 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     5221 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     5281 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     5341 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     5401 cgtctatatc accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca
     5461 caacatcgag gacggcggcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     5521 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc taccagtcca agctgagcaa
     5581 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     5641 cactctcggc atggacgagc tgtacaaggg aagcggagct actaacttca gcctgctgaa
     5701 gcaggctgga gacgtggagg agaaccctgg acctgactat aaggaccacg acggagacta
     5761 caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg
     5821 gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat
     5881 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     5941 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     6001 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     6061 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     6121 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     6181 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     6241 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     6301 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     6361 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     6421 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     6481 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     6541 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     6601 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     6661 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     6721 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     6781 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     6841 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     6901 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     6961 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     7021 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     7081 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     7141 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     7201 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     7261 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     7321 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     7381 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     7441 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     7501 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     7561 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     7621 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     7681 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     7741 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     7801 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     7861 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     7921 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     7981 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     8041 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     8101 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     8161 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     8221 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     8281 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     8341 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     8401 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     8461 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     8521 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     8581 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     8641 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     8701 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     8761 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     8821 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     8881 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     8941 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     9001 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     9061 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     9121 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     9181 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     9241 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     9301 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     9361 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     9421 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     9481 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     9541 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     9601 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     9661 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     9721 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     9781 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     9841 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     9901 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc
     9961 ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag taagaattcc tagagctcgc
    10021 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
    10081 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
    10141 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
    10201 aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgcg actctagatc
    10261 ataatcagcc cgggggatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
    10321 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
    10381 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
    10441 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
    10501 tgggctctat ggaaccagct ggggcgcgat aacttcgtat aggagacttt atacgaagtt
    10561 atgcgccatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
    10621 ccatcacaaa gctctgacct caatcctata gaaaggagga atgagccaaa attcacccaa
    10681 cttattgtgg gaagcttgtg gaaggctact cgaaatgttt gacccaagtt aaacaattta
    10741 aaggcaatgc taccaaatac taattgagtg tatgttaact tctgacccac tgggaatgtg
    10801 atgaaagaaa taaaagctga aatgaatcat tctctctact attattctga tatttcacat
    10861 tcttaaaata aagtggtgat cctaactgac cttaagacag ggaatcttta ctcggattaa
    10921 atgtcaggaa ttgtgaaaaa gtgagtttaa atgtatttgg ctaaggtgta tgtaaacttc
    10981 cgacttcaac tgtaggggat cctctagagt cgacctgcag gcatgcaagc ttggcgtaat
    11041 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
    11101 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
    11161 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
    11221 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
    11281 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
    11341 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
    11401 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
    11461 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
    11521 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
    11581 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
    11641 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
    11701 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
    11761 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
    11821 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
    11881 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
    11941 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
    12001 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
    12061 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
    12121 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
    12181 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
    12241 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
    12301 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
    12361 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
    12421 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
    12481 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
    12541 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
    12601 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
    12661 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
    12721 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
    12781 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
    12841 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
    12901 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
    12961 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
    13021 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
    13081 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
    13141 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
    13201 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
    13261 ttcgtc
//
