LOCUS       sequence-330216-       13012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA (#2) to generate a knockout of human CRBN by targeting the 
            start 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 Dec 15, 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 (#2)
FEATURES             Location/Qualifiers
     source          1..13012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    56..173
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        224..472
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     224..244
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     395..414
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        493..568
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        630..841
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      860..869
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             866..4969
                     /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             866..4969
                     /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             4970..5017
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5018..5041
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5051..5107
                     /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             5108..5704
                     /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     5601..5621
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5720..6308
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5773..5793)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6191..6202)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6380..6613
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6639..6656)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6645..6869
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6915..7343
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7002..7021)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7212..7233
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7352..7372)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7357..7686
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7537..7672
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7599..7618
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7734..7781
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7800..8174
                     /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    8304..8425
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8341..8360)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8395..8414
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8474..8490)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8474..8490)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8487..8509)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8498..8514
                     /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(8522..8528,8529..8546,8547..8552))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8567..8588
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8705..8722)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8876..9464)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8956..8975)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9635..10426,10427..10495))
                     /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     10258..10277
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10496..10600)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10675..10694)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10866..11245
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11247..11445
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11400..11420
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11463..11643
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11690..11815
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12308..12541
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12726..12770
                     /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 cggatcggca ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta
       61 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
      121 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
      181 attacaggga cagcagagat ccagtttggt taattaaggt accgagggcc tatttcccat
      241 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt
      301 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
      361 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
      421 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgcaggacg
      481 ctgcgcacaa cagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
      541 acttgaaaaa gtggcaccga gtcggtgctt ttttgaattc gctagctagg tcttgaaagg
      601 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
      661 gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa
      721 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
      781 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
      841 ggaccggttc tagagcgctg ccaccatgga caagaagtac agcatcggcc tggacatcgg
      901 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
      961 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     1021 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     1081 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     1141 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     1201 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     1261 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     1321 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     1381 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     1441 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     1501 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     1561 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     1621 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     1681 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     1741 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     1801 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     1861 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     1921 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     1981 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     2041 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     2101 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     2161 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     2221 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     2281 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     2341 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     2401 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     2461 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     2521 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     2581 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     2641 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     2701 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     2761 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     2821 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     2881 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     2941 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     3001 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     3061 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     3121 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     3181 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     3241 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     3301 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     3361 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     3421 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     3481 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     3541 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     3601 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     3661 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     3721 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     3781 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     3841 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     3901 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     3961 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     4021 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     4081 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     4141 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     4201 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     4261 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     4321 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     4381 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     4441 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     4501 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     4561 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     4621 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     4681 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     4741 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     4801 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     4861 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     4921 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca agcgacctgc
     4981 cgccacaaag aaggctggac aggctaagaa gaagaaagat tacaaagacg atgacgataa
     5041 gggatccggc gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc
     5101 tggaccgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     5161 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     5221 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     5281 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     5341 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     5401 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     5461 ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct
     5521 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     5581 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     5641 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     5701 ctgaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     5761 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     5821 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     5881 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     5941 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     6001 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     6061 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     6121 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     6181 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     6241 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     6301 ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     6361 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     6421 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     6481 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     6541 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     6601 aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     6661 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     6721 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     6781 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     6841 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     6901 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     6961 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     7021 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     7081 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     7141 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     7201 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     7261 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     7321 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     7381 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     7441 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     7501 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     7561 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     7621 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     7681 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca
     7741 attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca
     7801 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
     7861 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     7921 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     7981 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     8041 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     8101 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     8161 aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     8221 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     8281 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     8341 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     8401 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     8461 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     8521 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     8581 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     8641 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     8701 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     8761 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     8821 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     8881 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     8941 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     9001 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     9061 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     9121 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     9181 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     9241 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     9301 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     9361 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     9421 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     9481 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     9541 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     9601 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     9661 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     9721 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     9781 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     9841 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     9901 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     9961 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
    10021 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
    10081 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
    10141 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
    10201 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
    10261 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
    10321 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
    10381 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
    10441 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
    10501 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
    10561 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
    10621 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
    10681 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
    10741 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
    10801 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
    10861 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
    10921 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
    10981 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
    11041 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
    11101 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
    11161 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
    11221 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
    11281 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
    11341 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
    11401 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt tttgcctgta
    11461 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
    11521 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
    11581 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
    11641 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg
    11701 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac
    11761 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat
    11821 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa
    11881 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
    11941 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc
    12001 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
    12061 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga
    12121 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag
    12181 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac
    12241 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag
    12301 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag
    12361 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga
    12421 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca
    12481 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc
    12541 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta
    12601 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca
    12661 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc
    12721 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt
    12781 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg
    12841 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt
    12901 caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa
    12961 tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg aa
//
