LOCUS       sequence-243741-       13204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral plasmid encoding Cas9, GFP, and a gRNA 
            backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13204)
  AUTHORS   Giuliano CJ, Lin A, Girish V, Sheltzer JM
  TITLE     Generating Single Cell-Derived Knockout Clones in Mammalian Cells 
            with CRISPR/Cas9.
  JOURNAL   Curr Protoc Mol Biol. 2019 Sep;128(1):e100. doi: 10.1002/cpmb.100.
  PUBMED    31503414
REFERENCE   2  (bases 1 to 13204)
  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: "Curr Protoc
            Mol Biol."; date: "2019-09"; volume: "128(1):e100. doi"; pages: " 
            10.1002/cpmb.100"
COMMENT     Sequence Label: Lenti-Cas9-gRNA-GFP
FEATURES             Location/Qualifiers
     source          1..13204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    93..114
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(129..134,135..152,153..159)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    167..183
                     /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     172..194
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     191..207
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     191..207
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     226..246
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        228..246
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        274..500
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             501..681
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    728..853
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1346..1579
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     promoter        complement(3618..3866)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3676..3695)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3846..3866)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3873..3990
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4059..4270
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4290..4299
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4299..4322
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4329..4349
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4374..8474
                     /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             4374..8474
                     /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             8475..8522
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8538..8594
                     /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             join(8595..8597,8598..8600,8601..9314)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(8595..8597,8598..8600,8601..9311)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8901..8920)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9248..9269
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    9330..9918
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9383..9403)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9801..9812)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9990..10223
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    10301..10422
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10338..10357)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10392..10411
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10462..10597
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10617..10636)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10618..10636)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10646..10663)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10646..10662)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10655..10677)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10804..11259
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10891..10910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11101..11122
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11285..11389
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11390..11458,11459..12250)
                     /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(11608..11627)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12421..13009
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12910..12929
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13163..13180
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
       61 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
      121 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      181 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
      241 taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt
      301 cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg
      361 catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg
      421 gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt
      481 gcctagctcg atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct
      541 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
      601 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
      661 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
      721 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
      781 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
      841 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
      901 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
      961 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     1021 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     1081 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     1141 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga
     1201 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     1261 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     1321 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     1381 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     1441 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     1501 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     1561 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcgcc atttgtctca
     1621 agatctagaa ttcaaaaaaa gcaccgactc ggtgccactt tgctgtttcc agcaaagttg
     1681 ataacggact agccttattt aaacttgcta tgctgtttcc agcatagctc ttaaacagag
     1741 acgtacaaaa aagagcaaga agctaaaaaa gatttaaaaa ttatttttag cgcagttaat
     1801 ggaacaggaa ctaaatttac cccaaaaata ttacgtgaat caggatataa cgttattgag
     1861 gttgaagagc atgcatttga agatgaaaca tttaaaaatg ttgtaaatcc aaatccagaa
     1921 tttgatcctg catgaaaaat accgcttgaa tatggtatta aacatgatgc agatattatt
     1981 attatgaatg acccagatgc tgacagattt ggaatggcaa taaaacatga tggtcatttt
     2041 gtaagattag atggaaatca aacaggacca attttaattg attgaaaatt atcaaatcta
     2101 aaacgcttaa atagcattcc aaaaaatccg gctctatatt caagttttgt aacaagtgat
     2161 ttgggtgata gaatcgctca tgaaaaatat ggagttaata ttgtaaaaac tttaactgga
     2221 tttaaatgaa tgggtagaga aattgctaaa gaagaagata acggattaaa ttttgttttt
     2281 gcttatgaag aaagttatgg atatgtaatt gatgactcag ctagagataa agatggaata
     2341 caagcttcta tattaatagc agaggctgct tgattttata aaaaacaaaa taaaacatta
     2401 gtagactatt tagaagattt atttaaagaa atgggtgcat attacacttt cactttaaac
     2461 ttgaatttta aaccagaaga aaagaaatta aaaattgaac cattaatgaa atcattgaga
     2521 gcaacaccct taactcaaat tgctggactt aaagttgtta atgttgaaga ctacatcgat
     2581 ggaatgtata atatgccagg acaagactta ctaaaatttt atttagaaga taagtcatga
     2641 tttgctgttc gcccaagtgg aactgaacct aaactaaaaa tttattttat aggtgttggt
     2701 gaatctgttc aaaacgctaa agttaaagta gacgaaatta ttaaagaatt aaaattaaaa
     2761 atgaatatat aggagaaaaa atgaaactaa acaaatatat agatcacaca ttattaaaac
     2821 aagatgctac gaaagctgaa attaaacaat tatgtgatga agcaattgaa tttgattttg
     2881 caacagtttg tgttaattca tattgaacaa gctattgtaa agaattatta aaaggcacaa
     2941 atgtaggaat aacaaatgtt gtaggttttc ctctaggtgc atgcacaaca gctacaaaag
     3001 cattcgaagt ttctgaagca attaaagatg gtgcaacaga aattgatatg gtattaaata
     3061 ttggtgcatt aaaagacaaa aattatgaat tagttttaga agacatgaaa gctgtaaaaa
     3121 aagcagctgg atcacatgtt gttaaatgta ttatggaaaa ttgtttatta acaaaagaag
     3181 aaatcatgaa agcttgtgaa atagctgttg aagctggatt agaatttgtt aaaacatcaa
     3241 caggattttc aaaatcaggt gcaacatttg aagatgttaa actaatgaag tcagttgtta
     3301 aagacaatgc tttagttaaa gcagctggtg gagttagaac atttgaagat gctcaaaaaa
     3361 tgattgaagc aggagctgac cgcttaggaa caagtggtgg agtagctatt attaaaggtg
     3421 aagaaaacaa cgcgagttac taaaactagc gtttttttat tttgctcatt tttattaaaa
     3481 gtttgcaaaa aggaacataa aaattctaat tattgatact aaagttatta aaaagaagat
     3541 tttggttgat tttataaagg tcatagaata taatatttta gcatgtgtat tttgtgtgct
     3601 catttacaac cgtctccggt gtttcgtcct ttccacaaga tatataaagc caagaaatcg
     3661 aaatactttc aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact
     3721 gcaaactacc caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg
     3781 tttacagtca aattaattct aattatctct ctaacagcct tgtatcgtat atgcaaatat
     3841 gaaggaatca tgggaaatag gccctcccac aattttaaaa gaaaaggggg gattgggggg
     3901 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     3961 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     4021 ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     4081 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
     4141 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     4201 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     4261 cagaacacag gtgtcgtgac gcgggatccg ccaccatgga ttacaaagac gatgacgata
     4321 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga
     4381 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg
     4441 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     4501 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     4561 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     4621 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     4681 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     4741 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     4801 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     4861 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     4921 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     4981 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     5041 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     5101 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     5161 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     5221 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     5281 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     5341 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     5401 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     5461 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     5521 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     5581 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     5641 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     5701 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     5761 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     5821 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     5881 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     5941 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     6001 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     6061 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     6121 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     6181 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     6241 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     6301 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     6361 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     6421 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     6481 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     6541 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     6601 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     6661 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     6721 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     6781 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     6841 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     6901 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     6961 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     7021 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     7081 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     7141 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     7201 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     7261 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     7321 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     7381 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     7441 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     7501 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     7561 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     7621 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     7681 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     7741 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     7801 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     7861 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     7921 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     7981 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     8041 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     8101 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     8161 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     8221 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     8281 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     8341 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     8401 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     8461 agctgggagg cgacaagcgt cctgctgcta ctaagaaagc tggtcaagct aagaaaaaga
     8521 aagctagcgg cagcggcgcc accaacttca gcctgctgaa gcaggccggc gacgtggagg
     8581 agaaccccgg ccccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     8641 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     8701 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     8761 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     8821 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     8881 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     8941 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     9001 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     9061 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     9121 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     9181 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     9241 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     9301 agctgtacaa gtagacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa
     9361 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     9421 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     9481 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     9541 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     9601 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     9661 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     9721 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     9781 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     9841 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     9901 tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct
     9961 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
    10021 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
    10081 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
    10141 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
    10201 gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta ttattcagta
    10261 tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta
    10321 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
    10381 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct
    10441 atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
    10501 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
    10561 ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct
    10621 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
    10681 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
    10741 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
    10801 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
    10861 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
    10921 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
    10981 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
    11041 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
    11101 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
    11161 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
    11221 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
    11281 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
    11341 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
    11401 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
    11461 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
    11521 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
    11581 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
    11641 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
    11701 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
    11761 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
    11821 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
    11881 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
    11941 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
    12001 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
    12061 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
    12121 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
    12181 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
    12241 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
    12301 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
    12361 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
    12421 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
    12481 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
    12541 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
    12601 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
    12661 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
    12721 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
    12781 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
    12841 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
    12901 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
    12961 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
    13021 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
    13081 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
    13141 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
    13201 acgc
//
