LOCUS       sequence-226805-       14059 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one expression vector for Cas9 and gRNA against CD44.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14059)
  TITLE     Yusa CRISPR-Cas9 and mammalian expression plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 14059)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pKLV2-U6(gCD44v2)-EF1a-BFP-Puro-Cas9(FZ)
FEATURES             Location/Qualifiers
     source          1..14059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        380..1561
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          611..1553
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1510..1530
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1574..1583
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1580..2281
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MGSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     CDS             2300..2353
                     /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             2354..2950
                     /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(2354..2373)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2850..2870
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2969..3022
                     /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             3026..3091
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3098..3118
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3143..7243
                     /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             3143..7243
                     /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             7244..7291
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(7354..7374)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7772..7783)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8235..8252)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8241..8465
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(8491..8511)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8491..8509)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8530..8546)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8530..8546)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8543..8565)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8554..8570
                     /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(8578..8584,8585..8602,8603..8608))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8623..8644
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8761..8778)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8932..9520)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9012..9031)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9691..10482,10483..10551))
                     /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     10314..10333
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10552..10656)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(10682..11137)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10819..10840)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     11031..11050
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11264..11286
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     11278..11295
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     11279..11295
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11305..11324
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        11305..11323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        11639..11837
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11792..11812
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11855..12035
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    12082..12207
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12706..12939
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             13124..13168
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    13466..13583
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        13658..13906
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     13658..13678
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     13829..13848
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 accccgggct gggctgagac ccgcagagga agacgctcta gggatttgtc ccggactagc
       61 gagatggcaa ggctgaggac gggaggctga ttgagaggcg aaggtacacc ctaatctcaa
      121 tacaaccttt ggagctaagc cagcaatggt agagggaaga ttctgcacgt cccttccagg
      181 cggcctcccc gtcaccaccc cccccaaccc gccccgaccg gagctgagag taattcatac
      241 aaaaggactc gcccctgcct tggggaatcc cagggaccgt cgttaaactc ccactaacgt
      301 agaacccaga gatcgctgcg ttcccgcccc ctcacccgcc cgctctcgtc atcactgagg
      361 tggagaagag catgcgtgag gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
      421 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaagtggcg
      481 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
      541 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
      601 cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg
      661 cccttgcgtg ccttgaatta cttccacgcc cctggctgca gtacgtgatt cttgatcccg
      721 agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc
      781 ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc
      841 accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac
      901 ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca
      961 ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat
     1021 gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag
     1081 ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg
     1141 caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg
     1201 ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca
     1261 cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc
     1321 gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt
     1381 ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta
     1441 ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt
     1501 ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt
     1561 ggaagcttgg cgcgccacca tgggcagcga gctgattaag gagaacatgc acatgaagct
     1621 gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa
     1681 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
     1741 cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac
     1801 ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt
     1861 caccacatac gaggacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     1921 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat
     1981 gcagaagaaa acactcggct gggaggcctt caccgagacc ctgtaccccg ctgacggcgg
     2041 cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa
     2101 catcaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta
     2161 ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca
     2221 gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa
     2281 tggagggggc agcggcgggg aaggaagggg ctccctgctc acctgcggcg acgtggaaga
     2341 gaaccccgga cctatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     2401 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     2461 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     2521 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     2581 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     2641 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     2701 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtgtcgcccg accaccaggg
     2761 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     2821 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     2881 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     2941 gcccggtgcc ggcggcgggt ccggaggaga gggcagagga agtctcctaa catgcggtga
     3001 cgtggaggag aatcctggcc caatggacta taaggaccac gacggagact acaaggatca
     3061 tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg
     3121 tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa
     3181 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt
     3241 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga
     3301 cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag
     3361 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga
     3421 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga
     3481 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac
     3541 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat
     3601 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct
     3661 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca
     3721 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc
     3781 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa
     3841 gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag
     3901 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga
     3961 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa
     4021 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa
     4081 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct
     4141 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca
     4201 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa
     4261 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa
     4321 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat
     4381 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa
     4441 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc
     4501 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac
     4561 cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg
     4621 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct
     4681 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat
     4741 gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa
     4801 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg
     4861 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata
     4921 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga
     4981 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga
     5041 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg
     5101 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca
     5161 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat
     5221 gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc
     5281 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa
     5341 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca
     5401 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca
     5461 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca
     5521 gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta
     5581 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc
     5641 cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa
     5701 caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga
     5761 ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca
     5821 gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc
     5881 cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat
     5941 cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa
     6001 agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa
     6061 agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg
     6121 aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa
     6181 ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc
     6241 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa
     6301 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg
     6361 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat
     6421 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag
     6481 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt
     6541 cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc
     6601 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt
     6661 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct
     6721 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct
     6781 ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa
     6841 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca
     6901 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag
     6961 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta
     7021 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac
     7081 cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa
     7141 gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg
     7201 cctgtacgag acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac
     7261 gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagcggcc gctcaacctc tggattacaa
     7321 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     7381 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     7441 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     7501 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     7561 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     7621 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     7681 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     7741 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     7801 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     7861 tcggatctcc ctttgggccg cctccccgca ggtaccttta agaccaatga cttacaaggc
     7921 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     7981 ccaaagaagt caagatctgc tttttgcctg tactgggtct ctctggttag accagagtct
     8041 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     8101 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     8161 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagttta
     8221 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     8281 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     8341 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     8401 ggacagcaag ggggaggatt gggaagtcaa tagcaggcat gctggggatg cggtgggctc
     8461 tatgggcggc cgttaattaa gcttttgttc cctttagtga gggttaattg cgcgcttggc
     8521 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     8581 catacgagcc gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     8641 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     8701 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     8761 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     8821 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     8881 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     8941 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     9001 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     9061 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     9121 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     9181 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     9241 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     9301 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     9361 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     9421 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     9481 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     9541 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     9601 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     9661 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     9721 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     9781 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     9841 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     9901 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     9961 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
    10021 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
    10081 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
    10141 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
    10201 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
    10261 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
    10321 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
    10381 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
    10441 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
    10501 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
    10561 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
    10621 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
    10681 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
    10741 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
    10801 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
    10861 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
    10921 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
    10981 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
    11041 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
    11101 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
    11161 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
    11221 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
    11281 aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt gactagtaca
    11341 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
    11401 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
    11461 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
    11521 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
    11581 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
    11641 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
    11701 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
    11761 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
    11821 gaggtctata taagcagcgc gttttgcctg tactgggtct ctctggttag accagatctg
    11881 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
    11941 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
    12001 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa
    12061 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg
    12121 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag
    12181 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg
    12241 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
    12301 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc
    12361 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga
    12421 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac
    12481 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag
    12541 caagcggccg gccgcgctga tcttcagacc tggaggagga gatatgaggg acaattggag
    12601 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
    12661 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
    12721 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
    12781 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
    12841 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
    12901 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
    12961 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
    13021 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
    13081 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt
    13141 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
    13201 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
    13261 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
    13321 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag
    13381 agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc caattctgca
    13441 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
    13501 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
    13561 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttggt
    13621 tagtaccggg ccctacgcgt ccaaggtcgg gcaggaagag ggcctatttc ccatgattcc
    13681 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
    13741 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
    13801 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
    13861 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggga ggtgttggac
    13921 gtgacggttt aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt
    13981 atcaacttga aaaagtggca ccgagtcggt gcttttttta cgcgtgaggg gcgcggaagc
    14041 cggggcggag gaggagcca
//
