LOCUS       sequence-90023-e       11054 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral Vector for SpCas9 Expression without sgRNA, Blasticidin 
            resistance, EFS Promoter driven.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11054)
  AUTHORS   Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey 
            ME, Thielke A, Aster JC, Regev A, Ebert BL
  TITLE     Generation of mouse models of myeloid malignancy with combinatorial 
            genetic lesions using CRISPR-Cas9 genome editing.
  JOURNAL   Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951.
  PUBMED    24952903
REFERENCE   2  (bases 1 to 11054)
  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: "Nat 
            Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951."
COMMENT     Sequence Label: pLKO5d.EFS.SpCas9.P2A.BSD
FEATURES             Location/Qualifiers
     source          1..11054
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        235..446
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      466..475
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             475..498
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             505..525
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             550..4650
                     /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             550..4650
                     /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             4651..4698
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4714..4770
                     /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             4771..5166
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5200..5788
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5253..5273)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5671..5682)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5860..6093
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6171..6292
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6208..6227)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6262..6281
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6332..6467
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6487..6506)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6488..6506)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6516..6533)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6516..6532)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6525..6547)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6674..7129
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6761..6780)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6971..6992
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7155..7259
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7260..7328,7329..8120)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7478..7497)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8291..8879
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8780..8799
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9033..9050
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9167..9188
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9203..9208,9209..9226,9227..9233)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9241..9257
                     /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)."
     primer_bind     9246..9268
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9265..9281
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9265..9281
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9300..9320
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9302..9320
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        9348..9574
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             9575..9755
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    9802..9927
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    10420..10653
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             10838..10882
                     /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"
ORIGIN
        1 aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggcgccgg ctcgaggcgg ccgctggctc cggtgcccgt cagtgggcag
      241 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg
      301 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
      361 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
      421 gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg gatccgccac catggattac
      481 aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc
      541 ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
      601 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
      661 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
      721 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
      781 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
      841 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
      901 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
      961 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     1021 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     1081 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     1141 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     1201 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     1261 ggcaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     1321 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     1381 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     1441 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     1501 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     1561 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     1621 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     1681 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     1741 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     1801 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     1861 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     1921 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     1981 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     2041 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     2101 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     2161 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     2221 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     2281 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     2341 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     2401 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     2461 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     2521 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     2581 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     2641 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     2701 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     2761 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     2821 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     2881 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     2941 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     3001 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     3061 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     3121 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     3181 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     3241 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     3301 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     3361 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     3421 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     3481 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     3541 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     3601 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     3661 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     3721 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     3781 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     3841 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     3901 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     3961 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     4021 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     4081 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     4141 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     4201 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     4261 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     4321 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     4381 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     4441 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     4501 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     4561 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     4621 cggatcgacc tgtctcagct gggaggcgac aagcgtcctg ctgctactaa gaaagctggt
     4681 caagctaaga aaaagaaagc tagcggcagc ggcgccacca acttcagcct gctgaagcag
     4741 gccggcgacg tggaggagaa ccccggcccc atggccaagc ctttgtctca agaagaatcc
     4801 accctcattg aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc
     4861 gtcgccagcg cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat
     4921 tttactgggg gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct
     4981 ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc
     5041 ggacggtgcc gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac
     5101 agtgatggac agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg
     5161 gagggcctgc agctgcagta gtaaacgcgt taagtcgaca atcaacctct ggattacaaa
     5221 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     5281 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     5341 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     5401 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     5461 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     5521 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     5581 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     5641 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     5701 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     5761 cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag
     5821 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     5881 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     5941 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     6001 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     6061 gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta
     6121 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     6181 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     6241 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     6301 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     6361 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     6421 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
     6481 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     6541 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     6601 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     6661 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     6721 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     6781 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     6841 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     6901 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     6961 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     7021 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     7081 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     7141 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     7201 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     7261 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     7321 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     7381 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     7441 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     7501 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     7561 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     7621 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     7681 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     7741 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     7801 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     7861 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     7921 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     7981 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     8041 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     8101 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     8161 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     8221 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     8281 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     8341 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     8401 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     8461 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     8521 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     8581 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     8641 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     8701 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     8761 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     8821 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     8881 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     8941 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     9001 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     9061 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     9121 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     9181 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     9241 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
     9301 caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc
     9361 aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga
     9421 gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg
     9481 aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga
     9541 tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg
     9601 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     9661 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     9721 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa
     9781 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg
     9841 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag
     9901 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg
     9961 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
    10021 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc
    10081 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga
    10141 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac
    10201 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag
    10261 caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga
    10321 attatataaa tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa
    10381 gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt
    10441 cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag
    10501 acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca
    10561 acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc
    10621 tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact
    10681 catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat
    10741 ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat
    10801 acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga
    10861 attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat
    10921 aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact
    10981 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc
    11041 aaccccgagg ggac
//
