LOCUS       sequence-330215-       13012 bp    DNA     circular SYN 14-APR-2025
DEFINITION  sgRNA (#1) to generate a knockout of human CRBN by targeting the 
            middle region of Exon1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13012)
  AUTHORS   Lier S, Sellmer A, Orben F, Heinzlmeir S, Krauss L, Schneeweis C, 
            Hassan Z, Schneider C, Schafer A, Pongratz H, Engleitner T, Ollinger
            R, Kuisl A, Bassermann F, Schlag C, Kong B, Dove S, Kuster B, Rad R,
            Reichert M, Wirth M, Saur D, Mahboobi S, Schneider G
  TITLE     A novel Cereblon E3 ligase modulator with antitumor activity in 
            gastrointestinal cancer.
  JOURNAL   Bioorg Chem. 2021 Nov 20:105505. doi: 10.1016/j.bioorg.2021.105505.
  PUBMED    34838332
REFERENCE   2  (bases 1 to 13012)
  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: "Bioorg 
            Chem. 2021 Nov 20:105505. doi: 10.1016/j.bioorg.2021.105505."
COMMENT     Sequence Label: lentiCRISPR v2 puro - CRBN (#1)
FEATURES             Location/Qualifiers
     source          1..13012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    145..262
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        313..561
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     313..333
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     484..503
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        582..657
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        719..930
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      949..958
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             955..5058
                     /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             955..5058
                     /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             5059..5106
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5107..5130
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5140..5196
                     /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             5197..5793
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     5690..5710
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5809..6397
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5862..5882)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6280..6291)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6469..6702
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6728..6745)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6734..6958
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7004..7432
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7091..7110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7301..7322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7441..7461)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7446..7775
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7626..7761
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7688..7707
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7823..7870
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7889..8263
                     /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    8393..8514
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8430..8449)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8484..8503
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8563..8579)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8563..8579)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8576..8598)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8587..8603
                     /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(8611..8617,8618..8635,8636..8641))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8656..8677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8794..8811)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8965..9553)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9045..9064)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9724..10515,10516..10584))
                     /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     10347..10366
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10585..10689)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10764..10783)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10955..11334
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11336..11534
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11489..11509
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11552..11732
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11779..11904
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12397..12630
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12815..12859
                     /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"
ORIGIN
        1 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga
       61 gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag
      121 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
      181 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
      241 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt
      301 aattaaggta ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag
      361 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
      421 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
      481 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
      541 ttgtggaaag gacgaaacac cggccggcga aggagatcag cgttttagag ctagaaatag
      601 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      661 tttgaattcg ctagctaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg
      721 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgatcc
      781 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
      841 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
      901 tttcgcaacg ggtttgccgc cagaacacag gaccggttct agagcgctgc caccatggac
      961 aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc
     1021 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
     1081 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
     1141 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
     1201 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
     1261 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
     1321 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
     1381 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
     1441 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
     1501 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
     1561 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
     1621 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt
     1681 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
     1741 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
     1801 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
     1861 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
     1921 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
     1981 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
     2041 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
     2101 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
     2161 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
     2221 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
     2281 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
     2341 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
     2401 gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg
     2461 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
     2521 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
     2581 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
     2641 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
     2701 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
     2761 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
     2821 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
     2881 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
     2941 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
     3001 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
     3061 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
     3121 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
     3181 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
     3241 atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag
     3301 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
     3361 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
     3421 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg
     3481 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
     3541 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
     3601 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
     3661 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
     3721 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
     3781 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
     3841 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
     3901 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
     3961 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
     4021 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
     4081 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
     4141 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
     4201 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
     4261 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
     4321 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
     4381 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
     4441 ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
     4501 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
     4561 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
     4621 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
     4681 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
     4741 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
     4801 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
     4861 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
     4921 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
     4981 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
     5041 tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga aggctggaca ggctaagaag
     5101 aagaaagatt acaaagacga tgacgataag ggatccggcg caacaaactt ctctctgctg
     5161 aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc cacggtgcgc
     5221 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     5281 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     5341 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     5401 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     5461 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     5521 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     5581 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     5641 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     5701 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     5761 acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg
     5821 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     5881 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     5941 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     6001 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     6061 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     6121 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     6181 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     6241 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     6301 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     6361 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt
     6421 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     6481 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     6541 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     6601 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     6661 gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt ttaaacccgc
     6721 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     6781 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     6841 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     6901 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     6961 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     7021 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     7081 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     7141 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     7201 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     7261 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     7321 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     7381 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     7441 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     7501 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     7561 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     7621 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     7681 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     7741 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     7801 ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
     7861 ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct
     7921 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
     7981 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag
     8041 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
     8101 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
     8161 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
     8221 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt
     8281 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     8341 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     8401 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     8461 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     8521 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     8581 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     8641 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     8701 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     8761 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     8821 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     8881 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     8941 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     9001 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     9061 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     9121 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     9181 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     9241 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     9301 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     9361 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     9421 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     9481 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     9541 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     9601 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     9661 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     9721 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     9781 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     9841 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     9901 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     9961 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
    10021 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
    10081 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
    10141 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
    10201 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
    10261 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
    10321 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
    10381 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
    10441 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
    10501 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
    10561 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
    10621 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
    10681 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
    10741 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
    10801 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
    10861 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
    10921 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
    10981 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
    11041 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
    11101 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
    11161 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
    11221 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
    11281 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
    11341 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
    11401 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
    11461 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
    11521 gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc agatctgagc
    11581 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
    11641 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
    11701 acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg
    11761 aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca
    11821 agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag
    11881 gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa
    11941 aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca
    12001 agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt
    12061 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
    12121 ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc
    12181 aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa
    12241 gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt
    12301 atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag
    12361 aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt
    12421 gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca
    12481 attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca
    12541 gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt
    12601 ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat
    12661 ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg
    12721 gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca
    12781 ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt
    12841 agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa
    12901 attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc
    12961 tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc ac
//
