LOCUS       sequence-90017-e       13729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral CRISPR-Cas9 delivery for SpCas9 and sgRNA. Coexpresses 
            tagRFP via P2A cleavage site. SFFV Promoter driven .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13729)
  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 13729)
  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: pL-CRISPR.SFFV.tRFP
FEATURES             Location/Qualifiers
     source          1..13729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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"
     promoter        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3847..3922
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3970..4087
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4145..4552
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      4552..4561
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4561..4584
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4591..4611
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4636..8736
                     /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             4636..8736
                     /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             8737..8784
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8800..8856
                     /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             8857..9570
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    9586..10174
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9639..9659)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10057..10068)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10246..10479
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    10557..10678
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10594..10613)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10648..10667
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10718..10853
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10873..10892)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10874..10892)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10902..10919)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10902..10918)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10911..10933)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11060..11515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11147..11166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11357..11378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11541..11645
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11646..11714,11715..12506)
                     /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(11864..11883)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12677..13265
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13166..13185
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13419..13436
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13553..13574
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13589..13594,13595..13612,13613..13619)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13627..13643
                     /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     13632..13654
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13651..13667
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13651..13667
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13686..13706
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        13688..13706
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cggttgtaaa
     1981 tgagcacaca aaatacacat gctaaaatat tatattctat gacctttata aaatcaacca
     2041 aaatcttctt tttaataact ttagtatcaa taattagaat ttttatgttc ctttttgcaa
     2101 acttttaata aaaatgagca aaataaaaaa acgctagttt tagtaactcg cgttgttttc
     2161 ttcaccttta ataatagcta ctccaccact tgttcctaag cggtcagctc ctgcttcaat
     2221 cattttttga gcatcttcaa atgttctaac tccaccagct gctttaacta aagcattgtc
     2281 tttaacaact gacttcatta gtttaacatc ttcaaatgtt gcacctgatt ttgaaaatcc
     2341 tgttgatgtt ttaacaaatt ctaatccagc ttcaacagct atttcacaag ctttcatgat
     2401 ttcttctttt gttaataaac aattttccat aatacattta acaacatgtg atccagctgc
     2461 tttttttaca gctttcatgt cttctaaaac taattcataa tttttgtctt ttaatgcacc
     2521 aatatttaat accatatcaa tttctgttgc accatcttta attgcttcag aaacttcgaa
     2581 tgcttttgta gctgttgtgc atgcacctag aggaaaacct acaacatttg ttattcctac
     2641 atttgtgcct tttaataatt ctttacaata gcttgttcaa tatgaattaa cacaaactgt
     2701 tgcaaaatca aattcaattg cttcatcaca taattgttta atttcagctt tcgtagcatc
     2761 ttgttttaat aatgtgtgat ctatatattt gtttagtttc attttttctc ctatatattc
     2821 atttttaatt ttaattcttt aataatttcg tctactttaa ctttagcgtt ttgaacagat
     2881 tcaccaacac ctataaaata aatttttagt ttaggttcag ttccacttgg gcgaacagca
     2941 aatcatgact tatcttctaa ataaaatttt agtaagtctt gtcctggcat attatacatt
     3001 ccatcgatgt agtcttcaac attaacaact ttaagtccag caatttgagt taagggtgtt
     3061 gctctcaatg atttcattaa tggttcaatt tttaatttct tttcttctgg tttaaaattc
     3121 aagtttaaag tgaaagtgta atatgcaccc atttctttaa ataaatcttc taaatagtct
     3181 actaatgttt tattttgttt tttataaaat caagcagcct ctgctattaa tatagaagct
     3241 tgtattccat ctttatctct agctgagtca tcaattacat atccataact ttcttcataa
     3301 gcaaaaacaa aatttaatcc gttatcttct tctttagcaa tttctctacc cattcattta
     3361 aatccagtta aagtttttac aatattaact ccatattttt catgagcgat tctatcaccc
     3421 aaatcacttg ttacaaaact tgaatataga gccggatttt ttggaatgct atttaagcgt
     3481 tttagatttg ataattttca atcaattaaa attggtcctg tttgatttcc atctaatctt
     3541 acaaaatgac catcatgttt tattgccatt ccaaatctgt cagcatctgg gtcattcata
     3601 ataataatat ctgcatcatg tttaatacca tattcaagcg gtatttttca tgcaggatca
     3661 aattctggat ttggatttac aacattttta aatgtttcat cttcaaatgc atgctcttca
     3721 acctcaataa cgttatatcc tgattcacgt aatatttttg gggtaaattt agttcctgtt
     3781 ccattaactg cgctaaaaat aatttttaaa tcttttttag cttcttgctc ttttttgtac
     3841 gtctctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     3901 aaaagtggca ccgagtcggt gcttttttga attctagatc ttgagacaaa tggcagtatt
     3961 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     4021 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     4081 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagcgagc
     4141 tgcagtaacg ccattttgca aggcatggaa aaataccaaa ccaagaatag agaagttcag
     4201 atcaagggcg ggtacatgaa aatagctaac gttgggccaa acaggatatc tgcggtgagc
     4261 agtttcggcc ccggcccggg gccaagaaca gatggtcacc gcagtttcgg ccccggcccg
     4321 aggccaagaa cagatggtcc ccagatatgg cccaaccctc agcagtttct taagacccat
     4381 cagatgtttc caggctcccc caaggacctg aaatgaccct gcgccttatt tgaattaacc
     4441 aatcagcctg cttctcgctt ctgttcgcgc gcttctgctt cccgagctct ataaaagagc
     4501 tcacaacccc tcactcggcg cgccagtcct ccgacagact gagtcggatc cgccaccatg
     4561 gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac
     4621 ggagtcccag cagccgacaa gaagtacagc atcggcctgg acatcggcac caactctgtg
     4681 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
     4741 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
     4801 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
     4861 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
     4921 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
     4981 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
     5041 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
     5101 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
     5161 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
     5221 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
     5281 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
     5341 ctgttcggca acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
     5401 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
     5461 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
     5521 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
     5581 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
     5641 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
     5701 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
     5761 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
     5821 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
     5881 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
     5941 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
     6001 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
     6061 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
     6121 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
     6181 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
     6241 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
     6301 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
     6361 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
     6421 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
     6481 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
     6541 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
     6601 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
     6661 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
     6721 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
     6781 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
     6841 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
     6901 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
     6961 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
     7021 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
     7081 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
     7141 gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg
     7201 ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg
     7261 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
     7321 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
     7381 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
     7441 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
     7501 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
     7561 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
     7621 ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac
     7681 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
     7741 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
     7801 atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
     7861 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
     7921 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
     7981 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
     8041 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
     8101 ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag
     8161 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
     8221 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
     8281 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
     8341 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
     8401 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
     8461 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
     8521 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
     8581 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
     8641 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
     8701 gagacacgga tcgacctgtc tcagctggga ggcgacaagc gtcctgctgc tactaagaaa
     8761 gctggtcaag ctaagaaaaa gaaagctagc ggcagcggcg ccaccaactt cagcctgctg
     8821 aagcaggccg gcgacgtgga ggagaacccc ggccccatgg tgtctaaggg cgaagagctg
     8881 attaaggaga acatgcacat gaagctgtat atggagggca ccgtgaacaa ccaccacttc
     8941 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
     9001 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac
     9061 ggcagcagaa ccttcatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc
     9121 cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct
     9181 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
     9241 aacttcccat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccaacacc
     9301 gagatgctgt accccgctga cggcggcctg gaaggcagaa gcgacatggc cctgaagctc
     9361 gtgggcgggg gccacctgat ctgcaacttc aagaccacat acagatccaa gaaacccgct
     9421 aagaacctca agatgcccgg cgtctactat gtggaccaca gactggaaag aatcaaggag
     9481 gccgacaaag agacctacgt cgagcagcac gaggtggctg tggccagata ctgcgacctc
     9541 cctagcaaac tggggcacaa acttaattga acgcgttaag tcgacaatca acctctggat
     9601 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     9661 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     9721 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     9781 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     9841 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     9901 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     9961 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
    10021 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
    10081 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
    10141 acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca
    10201 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
    10261 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
    10321 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
    10381 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
    10441 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tacgtatagt agttcatgtc
    10501 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
    10561 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
    10621 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
    10681 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
    10741 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
    10801 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
    10861 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
    10921 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
    10981 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
    11041 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
    11101 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
    11161 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
    11221 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
    11281 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
    11341 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
    11401 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
    11461 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
    11521 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
    11581 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
    11641 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
    11701 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
    11761 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
    11821 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
    11881 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
    11941 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
    12001 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
    12061 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
    12121 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
    12181 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
    12241 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
    12301 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
    12361 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
    12421 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
    12481 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
    12541 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
    12601 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
    12661 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
    12721 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
    12781 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
    12841 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
    12901 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
    12961 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
    13021 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
    13081 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
    13141 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
    13201 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
    13261 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
    13321 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
    13381 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
    13441 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
    13501 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
    13561 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
    13621 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
    13681 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagc
//
