LOCUS       sequence-251949-       12838 bp    DNA     circular SYN 16-APR-2025
DEFINITION  Expresses human codon-optimized S. pyogenes Cas9 protein and 
            blasticidin resistance from EFS promoter. 3rd generation lentiviral 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12838)
  AUTHORS   Sanjana NE, Shalem O, Zhang F
  TITLE     Improved vectors and genome-wide libraries for CRISPR screening.
  JOURNAL   Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.
  PUBMED    25075903
REFERENCE   2  (bases 1 to 12838)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2014-08"; volume: "11"; issue: 
            "8"; pages: "783-4"
COMMENT     Sequence Label: lentiCas9-Blast
FEATURES             Location/Qualifiers
     source          1..12838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             119..299
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(325..342)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    331..555
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      601..1029
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(688..707)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     898..919
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1038..1058)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1203..1337
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1264..1283
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        1399..1446
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1465..1839
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1969..2090
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2006..2025)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2060..2079
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2139..2155)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2139..2155)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2152..2174)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2163..2179
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2187..2193,2194..2211,2212..2217))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2232..2253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2370..2387)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2541..3129)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2621..2640)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3300..4091,4092..4160))
                     /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     3923..3942
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4161..4265)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4340..4359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4531..4910
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4912..5110
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5065..5085
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5128..5308
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5355..5480
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5973..6206
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6391..6435
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    6733..6850
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6943..7154
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7173..7182
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7179..11282
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7179..11282
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             11283..11330
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             11331..11354
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             11364..11420
                     /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             11421..11819
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    11844..12432
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11897..11917)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12315..12326)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(12435..12451)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12436..12452)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag
       61 gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg
      121 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      181 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      241 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      301 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
      361 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
      421 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
      481 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
      541 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
      601 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      661 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      721 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      781 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      841 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      901 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      961 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1021 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     1081 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     1141 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     1201 agtcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     1261 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     1321 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     1381 cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     1441 tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc ggtgctcacc
     1501 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac
     1561 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg
     1621 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac
     1681 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg
     1741 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc
     1801 ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat
     1861 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     1921 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     1981 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     2041 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     2101 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     2161 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     2221 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     2281 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2341 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2401 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2461 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2521 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2581 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2641 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2701 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2761 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2821 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2881 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2941 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     3001 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3061 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3121 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3181 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3241 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3301 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3361 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3421 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3481 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3541 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3601 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3661 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3721 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3781 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3841 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3901 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3961 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4021 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4081 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4141 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4201 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4261 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     4321 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     4381 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     4441 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     4501 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
     4561 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     4621 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     4681 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     4741 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     4801 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     4861 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     4921 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     4981 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     5041 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     5101 atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat ctgagcctgg
     5161 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     5221 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     5281 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
     5341 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
     5401 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
     5461 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
     5521 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
     5581 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac
     5641 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat
     5701 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg
     5761 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg
     5821 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     5881 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     5941 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     6001 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     6061 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     6121 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     6181 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     6241 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     6301 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     6361 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     6421 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     6481 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     6541 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     6601 aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga
     6661 tccattcgat tagtgaacgg atcggcactg cgtgcgccaa ttctgcagac aaatggcagt
     6721 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     6781 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     6841 tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggttaa ttagctagct
     6901 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc
     6961 ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt
     7021 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     7081 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     7141 ccgccagaac acaggaccgg ttctagagcg ctgccaccat ggacaagaag tacagcatcg
     7201 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     7261 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     7321 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     7381 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     7441 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     7501 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     7561 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     7621 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     7681 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     7741 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     7801 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     7861 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     7921 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     7981 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     8041 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     8101 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     8161 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     8221 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     8281 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     8341 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     8401 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     8461 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     8521 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     8581 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     8641 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     8701 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     8761 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     8821 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     8881 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     8941 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     9001 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     9061 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     9121 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     9181 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     9241 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     9301 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     9361 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     9421 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     9481 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     9541 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     9601 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     9661 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     9721 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     9781 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     9841 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     9901 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     9961 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
    10021 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
    10081 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
    10141 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
    10201 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
    10261 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
    10321 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
    10381 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
    10441 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
    10501 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
    10561 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
    10621 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctactg gggatcacca
    10681 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
    10741 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
    10801 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
    10861 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
    10921 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
    10981 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
    11041 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
    11101 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
    11161 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
    11221 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
    11281 acaagcgacc tgccgccaca aagaaggctg gacaggctaa gaagaagaaa gattacaaag
    11341 acgatgacga taagggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg
    11401 tcgaagagaa tcctggaccg atggccaagc ctttgtctca agaagaatcc accctcattg
    11461 aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg
    11521 cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg
    11581 gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga
    11641 cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc
    11701 gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac
    11761 agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag
    11821 aattcgatat caagcttatc ggtaatcaac ctctggatta caaaatttgt gaaagattga
    11881 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
    11941 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
    12001 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
    12061 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
    12121 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
    12181 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
    12241 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
    12301 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
    12361 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
    12421 ccgcctcccc gcatcgatac cgtcgacctc gagacctaga aaaacatgga gcaatcacaa
    12481 gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg
    12541 aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg
    12601 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
    12661 gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc
    12721 agaactacac accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc
    12781 tagtaccagt tgagcaagag aaggtagaag aagccaatga aggagagaac acccgctt
//
