LOCUS       sequence-218584-       13013 bp    DNA     circular SYN 14-APR-2025
DEFINITION  CRISPR KO murine CLYBL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  AUTHORS   Shen H, Campanello GC, Flicker D, Grabarek Z, Hu J, Luo C, Banerjee 
            R, Mootha VK
  TITLE     The Human Knockout Gene CLYBL Connects Itaconate to Vitamin B12.
  JOURNAL   Cell. 2017 Nov 2;171(4):771-782.e11. doi: 
            10.1016/j.cell.2017.09.051. Epub 2017 Oct 19.
  PUBMED    29056341
REFERENCE   2  (bases 1 to 13013)
  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"; doi: 
            "10.1016/j.cell.2017.09.051"; journalName: "Cell"; date: "2017-11-2-
            2"; volume: "171"; issue: "4"; pages: "771-782.e11"
COMMENT     Sequence Label: MsCLYBL_sgRNA6
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /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    1606..1723
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1774..2022
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1774..1794
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1945..1964
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2044..2119
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2181..2392
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2411..2420
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2417..6520
                     /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             2417..6520
                     /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             6521..6568
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6569..6592
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6602..6658
                     /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             6659..7255
                     /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     7152..7172
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7271..7859
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7324..7344)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7742..7753)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7931..8164
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8190..8207)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8196..8420
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8466..8894
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8553..8572)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8763..8784
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8903..8923)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8908..9237
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9088..9223
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9150..9169
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9285..9332
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9351..9725
                     /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    9855..9976
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9892..9911)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9946..9965
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10025..10041)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10025..10041)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10038..10060)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10049..10065
                     /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(10073..10079,10080..10097,10098..10103))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10118..10139
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10256..10273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10427..11015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10507..10526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11186..11977,11978..12046))
                     /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     11809..11828
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12047..12151)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12226..12245)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12417..12796
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        12798..12996
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     12951..12971
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     1501 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     1561 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     1621 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     1681 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     1741 cagcagagat ccagtttggt taattaaggt accgagggcc tatttcccat gattccttca
     1801 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
     1861 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     1921 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     1981 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgcatagag cactgctctc
     2041 cgggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     2101 agtggcaccg agtcggtgct tttttgaatt cgctagctag gtcttgaaag gagtgggaat
     2161 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
     2221 gggaggggtc ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
     2281 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     2341 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggaccggtt
     2401 ctagagcgct gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaactc
     2461 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct
     2521 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag
     2581 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg
     2641 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga
     2701 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg
     2761 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat
     2821 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta
     2881 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
     2941 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct
     3001 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag
     3061 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa
     3121 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa
     3181 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct
     3241 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa
     3301 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc
     3361 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct
     3421 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag
     3481 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt
     3541 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag
     3601 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca
     3661 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga
     3721 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct
     3781 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
     3841 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat
     3901 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta
     3961 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag
     4021 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac
     4081 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt
     4141 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
     4201 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
     4261 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
     4321 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
     4381 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
     4441 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     4501 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     4561 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     4621 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     4681 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     4741 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     4801 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     4861 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     4921 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     4981 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     5041 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     5101 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     5161 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     5221 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     5281 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     5341 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     5401 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
     5461 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     5521 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     5581 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     5641 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     5701 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     5761 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     5821 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     5881 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     5941 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     6001 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     6061 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     6121 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     6181 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     6241 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     6301 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     6361 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
     6421 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     6481 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aagcgacctg ccgccacaaa
     6541 gaaggctgga caggctaaga agaagaaaga ttacaaagac gatgacgata agggatccgg
     6601 cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgac
     6661 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     6721 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     6781 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     6841 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     6901 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     6961 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     7021 gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     7081 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     7141 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     7201 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
     7261 ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     7321 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     7381 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     7441 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     7501 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     7561 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     7621 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     7681 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     7741 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     7801 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg
     7861 tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     7921 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     7981 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     8041 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     8101 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     8161 agcagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     8221 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     8281 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     8341 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     8401 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     8461 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     8521 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     8581 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     8641 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     8701 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     8761 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     8821 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     8881 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     8941 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     9001 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     9061 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     9121 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     9181 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     9241 ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat
     9301 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt
     9361 tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga
     9421 ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg
     9481 acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg
     9541 cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca
     9601 cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc
     9661 gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg
     9721 actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     9781 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     9841 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     9901 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     9961 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
    10021 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
    10081 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
    10141 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
    10201 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
    10261 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
    10321 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
    10381 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
    10441 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
    10501 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
    10561 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
    10621 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
    10681 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
    10741 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
    10801 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
    10861 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
    10921 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
    10981 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
    11041 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
    11101 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
    11161 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
    11221 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
    11281 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
    11341 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
    11401 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
    11461 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
    11521 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
    11581 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
    11641 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
    11701 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
    11761 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
    11821 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
    11881 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
    11941 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
    12001 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
    12061 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
    12121 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
    12181 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
    12241 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
    12301 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
    12361 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
    12421 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
    12481 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
    12541 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
    12601 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
    12661 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
    12721 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
    12781 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
    12841 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
    12901 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
    12961 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt act
//
