LOCUS       sequence-394138-       12824 bp    DNA     circular SYN 14-APR-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 Apr 14, 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 #4-Blast
FEATURES             Location/Qualifiers
     source          1..12824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(222..239)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    228..452
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      498..926
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(585..604)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     795..816
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(935..955)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        940..1269
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1120..1255
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1182..1201
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        1317..1364
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1383..1757
                     /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    1887..2008
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1924..1943)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1978..1997
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2057..2073)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2057..2073)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2070..2092)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2081..2097
                     /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(2105..2111,2112..2129,2130..2135))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2150..2171
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2288..2305)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2459..3047)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2539..2558)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3218..4009,4010..4078))
                     /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     3841..3860
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4079..4183)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4258..4277)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4449..4828
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4830..5028
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4983..5003
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5046..5226
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5273..5398
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5891..6124
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6309..6353
                     /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    6651..6768
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6819..7067
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6819..6839
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6990..7009
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7089..7164
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        7226..7437
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7456..7465
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7462..11565
                     /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             7462..11565
                     /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             11566..11613
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             11614..11637
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             11647..11703
                     /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             11704..12102
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    12127..12715
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12180..12200)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12598..12609)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             join(12787..12824,1..196)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
       61 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
      121 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
      181 tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
      241 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
      301 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
      361 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
      421 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
      481 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
      541 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
      601 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
      661 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
      721 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
      781 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
      841 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
      901 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
      961 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     1021 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     1081 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     1141 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     1201 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     1261 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg
     1321 acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa
     1381 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
     1441 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
     1501 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
     1561 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
     1621 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
     1681 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
     1741 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa
     1801 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     1861 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     1921 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     1981 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     2041 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2101 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2161 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2221 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2281 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2341 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2401 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2461 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2521 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2581 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2641 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2701 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2761 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2821 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2881 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2941 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3001 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3061 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3121 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3181 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3241 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3301 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3361 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3421 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3481 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3541 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3601 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3661 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3721 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3781 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3841 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3901 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3961 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4021 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4081 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4141 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4201 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     4261 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     4321 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     4381 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     4441 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     4501 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     4561 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     4621 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     4681 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     4741 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     4801 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     4861 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     4921 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     4981 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct
     5041 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     5101 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     5161 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     5221 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg
     5281 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag
     5341 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag
     5401 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa
     5461 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt
     5521 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc
     5581 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta
     5641 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga
     5701 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag
     5761 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
     5821 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
     5881 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
     5941 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
     6001 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
     6061 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
     6121 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg
     6181 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg
     6241 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc
     6301 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt
     6361 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct
     6421 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
     6481 attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag
     6541 gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat
     6601 cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa ttttaaaaga
     6661 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     6721 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     6781 agggacagca gagatccagt ttggttaatt aaggtaccga gggcctattt cccatgattc
     6841 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg
     6901 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     6961 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     7021 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgtg gaaagtagga
     7081 ttacccatgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     7141 gaaaaagtgg caccgagtcg gtgctttttt gaattcgcta gctaggtctt gaaaggagtg
     7201 ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     7261 ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg
     7321 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     7381 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggac
     7441 cggttctaga gcgctgccac catggacaag aagtacagca tcggcctgga catcggcacc
     7501 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     7561 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     7621 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     7681 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     7741 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     7801 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     7861 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     7921 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     7981 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     8041 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     8101 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     8161 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     8221 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     8281 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     8341 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     8401 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     8461 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     8521 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     8581 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     8641 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     8701 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     8761 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     8821 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     8881 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     8941 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     9001 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     9061 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     9121 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     9181 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     9241 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     9301 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     9361 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     9421 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     9481 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     9541 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     9601 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     9661 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     9721 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     9781 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     9841 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     9901 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     9961 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
    10021 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
    10081 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
    10141 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
    10201 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
    10261 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
    10321 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
    10381 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
    10441 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
    10501 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
    10561 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
    10621 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
    10681 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
    10741 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
    10801 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
    10861 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
    10921 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
    10981 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
    11041 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
    11101 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
    11161 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
    11221 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
    11281 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
    11341 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
    11401 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
    11461 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
    11521 ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaagcg acctgccgcc
    11581 acaaagaagg ctggacaggc taagaagaag aaagattaca aagacgatga cgataaggga
    11641 tccggcgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga gaatcctgga
    11701 ccgatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
    11761 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
    11821 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
    11881 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
    11941 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
    12001 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
    12061 attcgtgaat tgctgccctc tggttatgtg tgggagggct aagaattcga tatcaagctt
    12121 atcggtaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
    12181 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
    12241 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
    12301 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
    12361 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
    12421 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
    12481 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
    12541 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
    12601 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
    12661 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
    12721 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
    12781 ggggactgga agggctaatt cactcccaac gaagacaaga tctg
//
