LOCUS       sequence-220723-       14064 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 14064)
  TITLE     Yusa CRISPR-Cas9 and mammalian expression plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 14064)
  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            "Unpublished"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..14064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        960..1158
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1113..1133
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1176..1356
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1403..1528
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2027..2260
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2445..2489
                     /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    2787..2904
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2979..3227
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2979..2999
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3150..3169
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3760..4945
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3991..4937
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4894..4914
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4959..4968
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4965..5666
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MGSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     CDS             5685..5738
                     /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             5739..6335
                     /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(5739..5758)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6235..6255
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             6354..6407
                     /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             6411..6476
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6483..6503
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6528..10628
                     /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             6528..10628
                     /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             10629..10676
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(10739..10759)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11157..11168)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11620..11637)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11626..11850
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(11876..11896)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(11876..11894)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(11915..11931)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11915..11931)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11928..11950)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11939..11955
                     /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(11963..11969,11970..11987,11988..11993))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12008..12029
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12146..12163)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12317..12905)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12397..12416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13076..13867,13868..13936))
                     /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     13699..13718
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13937..14041)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgactagtac
      661 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      721 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      781 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      841 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      901 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      961 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     1021 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     1081 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     1141 ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct
     1201 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     1261 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     1321 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     1381 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     1441 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     1501 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     1561 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     1621 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     1681 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     1741 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     1801 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     1861 gcaagcggcc ggccgcgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     1921 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1981 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     2041 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     2101 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     2161 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     2221 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     2281 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     2341 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     2401 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     2461 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     2521 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     2581 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     2641 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     2701 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     2761 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     2821 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     2881 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     2941 ttagtaccgg gccctacgcg tccaaggtcg ggcaggaaga gggcctattt cccatgattc
     3001 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg
     3061 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     3121 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     3181 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg aggtgttgga
     3241 cgtgacggtt taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt
     3301 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt acgcgtgagg ggcgcggaag
     3361 ccggggcgga ggaggagcca accccgggct gggctgagac ccgcagagga agacgctcta
     3421 gggatttgtc ccggactagc gagatggcaa ggctgaggac gggaggctga ttgagaggcg
     3481 aaggtacacc ctaatctcaa tacaaccttt ggagctaagc cagcaatggt agagggaaga
     3541 ttctgcacgt cccttccagg cggcctcccc gtcaccaccc cccccaaccc gccccgaccg
     3601 gagctgagag taattcatac aaaaggactc gcccctgcct tggggaatcc cagggaccgt
     3661 cgttaaactc ccactaacgt agaacccaga gatcgctgcg ttcccgcccc ctcacccgcc
     3721 cgctctcgtc atcactgagg tggagaagag catgcgtgag gctccggtgc ccgtcagtgg
     3781 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc
     3841 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     3901 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     3961 tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct
     4021 ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacgcc cctggctgca
     4081 gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg
     4141 cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcttgggc gctggggccg
     4201 ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc
     4261 catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa
     4321 tgcgggccaa gatcgatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg
     4381 gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga
     4441 gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc
     4501 cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg
     4561 aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg
     4621 gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg
     4681 cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct
     4741 tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca
     4801 ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat
     4861 ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt
     4921 tttttcttcc atttcaggtg tcgtgggaag cttggcgcgc caccatgggc agcgagctga
     4981 ttaaggagaa catgcacatg aagctgtaca tggagggcac cgtggacaac catcacttca
     5041 agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg
     5101 tggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctactagc ttcctctacg
     5161 gcagcaagac cttcatcaac cacacccagg gcatccccga cttcttcaag cagtccttcc
     5221 ctgagggctt cacatgggag agagtcacca catacgagga cgggggcgtg ctgaccgcta
     5281 cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga
     5341 acttcacatc caacggccct gtgatgcaga agaaaacact cggctgggag gccttcaccg
     5401 agaccctgta ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg
     5461 tgggcgggag ccatctgatc gcaaacatca agaccacata tagatccaag aaacccgcta
     5521 agaacctcaa gatgcctggc gtctactatg tggactacag actggaaaga atcaaggagg
     5581 ccaacaacga gacctacgtc gagcagcacg aggtggcagt ggccagatac tgcgacctcc
     5641 ctagcaaact ggggcacaag cttaatggag ggggcagcgg cggggaagga aggggctccc
     5701 tgctcacctg cggcgacgtg gaagagaacc ccggacctat gaccgagtac aagcccacgg
     5761 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     5821 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     5881 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     5941 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     6001 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     6061 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     6121 tcggcgtgtc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     6181 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     6241 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     6301 cgcgcacctg gtgcatgacc cgcaagcccg gtgccggcgg cgggtccgga ggagagggca
     6361 gaggaagtct cctaacatgc ggtgacgtgg aggagaatcc tggcccaatg gactataagg
     6421 accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg
     6481 ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca
     6541 gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
     6601 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
     6661 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
     6721 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     6781 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     6841 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     6901 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     6961 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     7021 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     7081 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     7141 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     7201 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc
     7261 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     7321 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     7381 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     7441 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     7501 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     7561 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg
     7621 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     7681 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     7741 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     7801 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     7861 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     7921 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     7981 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     8041 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca
     8101 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     8161 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     8221 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     8281 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     8341 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     8401 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     8461 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     8521 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     8581 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     8641 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
     8701 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
     8761 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
     8821 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
     8881 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
     8941 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
     9001 aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct
     9061 ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca
     9121 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc
     9181 tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag
     9241 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
     9301 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
     9361 atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
     9421 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
     9481 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
     9541 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
     9601 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
     9661 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
     9721 acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag
     9781 tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
     9841 gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact
     9901 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
     9961 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
    10021 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
    10081 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
    10141 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
    10201 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
    10261 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc
    10321 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
    10381 atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg
    10441 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
    10501 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
    10561 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
    10621 gaggcgacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagtaag
    10681 cggccgctca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
    10741 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
    10801 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
    10861 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
    10921 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
    10981 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
    11041 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
    11101 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
    11161 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
    11221 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcaggtac
    11281 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
    11341 ggggactgga agggctaatt cactcccaaa gaagtcaaga tctgcttttt gcctgtactg
    11401 ggtctctctg gttagaccag agtctctctg gttagaccag atctgagcct gggagctctc
    11461 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
    11521 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
    11581 agtgtggaaa atctctagca gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
    11641 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
    11701 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
    11761 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gtcaatagca
    11821 ggcatgctgg ggatgcggtg ggctctatgg gcggccgtta attaagcttt tgttcccttt
    11881 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
    11941 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
    12001 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
    12061 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
    12121 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
    12181 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
    12241 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
    12301 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
    12361 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
    12421 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
    12481 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
    12541 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
    12601 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
    12661 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
    12721 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
    12781 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
    12841 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
    12901 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
    12961 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
    13021 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
    13081 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
    13141 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
    13201 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
    13261 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
    13321 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
    13381 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
    13441 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
    13501 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
    13561 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
    13621 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
    13681 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
    13741 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
    13801 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
    13861 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
    13921 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
    13981 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
    14041 gcacatttcc ccgaaaagtg ccac
//
