LOCUS       sequence-394137-       12824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9 and sgRNA guide targeting VRK1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12824)
  AUTHORS   So J, Mabe NW, Englinger B, Chow KH, Moyer SM, Yerrum S, Trissal MC,
            Marques JG, Kwon JJ, Shim B, Pal S, Panditharatna E, Quinn T, 
            Schaefer DA, Jeong D, Mayhew DL, Hwang J, Beroukhim R, Ligon KL, 
            Stegmaier K, Filbin MG, Hahn WC
  TITLE     VRK1 as a synthetic lethal target in VRK2 promoter-methylated 
            cancers of the nervous system.
  JOURNAL   JCI Insight. 2022 Oct 10;7(19):e158755. doi: 
            10.1172/jci.insight.158755.
  PUBMED    36040810
REFERENCE   2  (bases 1 to 12824)
  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: "JCI 
            Insight."; date: "2022-10-10"; volume: "7(19):e158755. doi"; pages: 
            " 10.1172/jci.insight.158755"
COMMENT     Sequence Label: LentiCas9-sgVRK1 #1-Blast
FEATURES             Location/Qualifiers
     source          1..12824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(207..224)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    213..437
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      483..911
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(570..589)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     780..801
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(920..940)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        925..1254
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1105..1240
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1167..1186
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        1302..1349
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1368..1742
                     /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    1872..1993
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1909..1928)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1963..1982
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2042..2058)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2042..2058)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2055..2077)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2066..2082
                     /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(2090..2096,2097..2114,2115..2120))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2135..2156
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2273..2290)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2444..3032)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2524..2543)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3203..3994,3995..4063))
                     /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     3826..3845
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4064..4168)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4243..4262)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4434..4813
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4815..5013
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4968..4988
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5031..5211
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5258..5383
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5876..6109
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6294..6338
                     /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    6636..6753
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6804..7052
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6804..6824
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6975..6994
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7074..7149
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        7211..7422
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7441..7450
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7447..11550
                     /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             7447..11550
                     /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             11551..11598
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             11599..11622
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             11632..11688
                     /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             11689..12087
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    12112..12700
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12165..12185)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12583..12594)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             join(12772..12824,1..181)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
      241 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
      301 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
      361 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
      421 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
      481 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
      541 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
      601 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
      661 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
      721 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
      781 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
      841 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
      901 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
      961 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     1021 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     1081 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     1141 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     1201 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     1261 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg
     1321 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga
     1381 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
     1441 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
     1501 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
     1561 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
     1621 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
     1681 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
     1741 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     1801 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     1861 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     1921 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     1981 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     2041 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     2101 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     2161 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2221 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     2281 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2341 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2401 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2461 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2521 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2581 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2641 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2701 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2761 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2821 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2881 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2941 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3001 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3061 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3121 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3181 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3241 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3301 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3361 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3421 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3481 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3541 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3601 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3661 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3721 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3781 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3841 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3901 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     3961 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4021 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4081 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4141 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     4201 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     4261 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     4321 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     4381 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
     4441 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     4501 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     4561 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     4621 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     4681 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     4741 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     4801 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     4861 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     4921 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     4981 taggcgtgta cggtgggagg tctatataag cagcgcgttt tgcctgtact gggtctctct
     5041 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     5101 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     5161 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
     5221 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
     5281 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
     5341 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
     5401 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
     5461 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
     5521 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     5581 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     5641 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     5701 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
     5761 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     5821 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     5881 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     5941 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     6001 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     6061 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     6121 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     6181 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     6241 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     6301 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     6361 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     6421 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     6481 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     6541 gtggagagag agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc
     6601 caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     6661 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     6721 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     6781 ccagtttggt taattaaggt accgagggcc tatttcccat gattccttca tatttgcata
     6841 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
     6901 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     6961 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     7021 ctttatatat cttgtggaaa ggacgaaaca ccgcccaata cttaggaaca cccgttttag
     7081 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     7141 agtcggtgct tttttgaatt cgctagctag gtcttgaaag gagtgggaat tggctccggt
     7201 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     7261 ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     7321 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     7381 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagagcgct
     7441 gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     7501 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     7561 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     7621 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     7681 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     7741 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     7801 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     7861 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     7921 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     7981 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     8041 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     8101 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     8161 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     8221 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     8281 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     8341 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     8401 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     8461 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     8521 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     8581 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     8641 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     8701 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     8761 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     8821 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     8881 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     8941 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     9001 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     9061 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     9121 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     9181 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     9241 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     9301 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     9361 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     9421 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     9481 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     9541 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     9601 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     9661 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     9721 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     9781 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     9841 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     9901 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     9961 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
    10021 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
    10081 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
    10141 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
    10201 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
    10261 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
    10321 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
    10381 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
    10441 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
    10501 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
    10561 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
    10621 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
    10681 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
    10741 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
    10801 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
    10861 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
    10921 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
    10981 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
    11041 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
    11101 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
    11161 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
    11221 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
    11281 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
    11341 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
    11401 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
    11461 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
    11521 cggatcgacc tgtctcagct gggaggcgac aagcgacctg ccgccacaaa gaaggctgga
    11581 caggctaaga agaagaaaga ttacaaagac gatgacgata agggatccgg cgcaacaaac
    11641 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgat ggccaagcct
    11701 ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc
    11761 atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact
    11821 ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact
    11881 gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg
    11941 ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc
    12001 aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg
    12061 ccctctggtt atgtgtggga gggctaagaa ttcgatatca agcttatcgg taatcaacct
    12121 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
    12181 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
    12241 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
    12301 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
    12361 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
    12421 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
    12481 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
    12541 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
    12601 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
    12661 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
    12721 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
    12781 taattcactc ccaacgaaga caagatctgc tttttgcttg tact
//
