LOCUS       sequence-218581-       13012 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 13012)
  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 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"; 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_sgRNA1
FEATURES             Location/Qualifiers
     source          1..13012
                     /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        2043..2118
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2180..2391
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2410..2419
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2416..6519
                     /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             2416..6519
                     /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             6520..6567
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6568..6591
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6601..6657
                     /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             6658..7254
                     /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     7151..7171
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7270..7858
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7323..7343)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7741..7752)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7930..8163
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8189..8206)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8195..8419
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8465..8893
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8552..8571)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8762..8783
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8902..8922)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8907..9236
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9087..9222
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9149..9168
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9284..9331
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9350..9724
                     /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    9854..9975
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9891..9910)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9945..9964
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10024..10040)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10024..10040)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10037..10059)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10048..10064
                     /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(10072..10078,10079..10096,10097..10102))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10117..10138
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10255..10272)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10426..11014)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10506..10525)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11185..11976,11977..12045))
                     /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     11808..11827
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12046..12150)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12225..12244)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12416..12795
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        12797..12995
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     12950..12970
                     /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 ccgcggaaca cggttcgtgg
     2041 aggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     2101 gtggcaccga gtcggtgctt ttttgaattc gctagctagg tcttgaaagg agtgggaatt
     2161 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     2221 ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     2281 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     2341 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggaccggttc
     2401 tagagcgctg ccaccatgga caagaagtac agcatcggcc tggacatcgg caccaactct
     2461 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
     2521 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
     2581 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
     2641 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     2701 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     2761 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     2821 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     2881 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     2941 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     3001 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     3061 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     3121 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     3181 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     3241 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     3301 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     3361 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     3421 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     3481 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
     3541 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     3601 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     3661 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     3721 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     3781 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
     3841 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
     3901 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
     3961 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
     4021 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
     4081 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
     4141 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
     4201 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
     4261 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
     4321 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
     4381 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
     4441 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
     4501 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
     4561 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     4621 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     4681 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     4741 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     4801 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     4861 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     4921 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     4981 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     5041 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     5101 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     5161 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     5221 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     5281 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     5341 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     5401 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     5461 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     5521 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     5581 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     5641 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     5701 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
     5761 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
     5821 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     5881 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     5941 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     6001 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     6061 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     6121 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     6181 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     6241 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     6301 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     6361 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
     6421 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     6481 tacgagacac ggatcgacct gtctcagctg ggaggcgaca agcgacctgc cgccacaaag
     6541 aaggctggac aggctaagaa gaagaaagat tacaaagacg atgacgataa gggatccggc
     6601 gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccgacc
     6661 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     6721 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     6781 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     6841 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     6901 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     6961 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     7021 tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     7081 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     7141 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     7201 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaacgcgt
     7261 taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     7321 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     7381 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     7441 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     7501 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     7561 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     7621 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     7681 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     7741 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     7801 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgt
     7861 cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     7921 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta
     7981 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     8041 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     8101 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     8161 gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     8221 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     8281 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     8341 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     8401 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     8461 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     8521 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     8581 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     8641 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     8701 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     8761 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     8821 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     8881 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     8941 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     9001 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     9061 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     9121 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     9181 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     9241 tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc
     9301 ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt
     9361 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac
     9421 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga
     9481 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc
     9541 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac
     9601 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg
     9661 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga
     9721 ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     9781 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     9841 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     9901 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     9961 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
    10021 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
    10081 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
    10141 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
    10201 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
    10261 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
    10321 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
    10381 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
    10441 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
    10501 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
    10561 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
    10621 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
    10681 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
    10741 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
    10801 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
    10861 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
    10921 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
    10981 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
    11041 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
    11101 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
    11161 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
    11221 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
    11281 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
    11341 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
    11401 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
    11461 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
    11521 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
    11581 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
    11641 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
    11701 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
    11761 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
    11821 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
    11881 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
    11941 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
    12001 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
    12061 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
    12121 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
    12181 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
    12241 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
    12301 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
    12361 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
    12421 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
    12481 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
    12541 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
    12601 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
    12661 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
    12721 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
    12781 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
    12841 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
    12901 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
    12961 ggtaggcgtg tacggtggga ggtctatata agcagcgcgt tttgcctgta ct
//
