LOCUS       sequence-327641-       11590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For CRISPR knockout of miR-451 by lentiviral delivery of Cas9 and 
            miR-451 gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11590)
  AUTHORS   Fang W, Bartel DP
  TITLE     MicroRNA Clustering Assists Processing of Suboptimal MicroRNA 
            Hairpins through the Action of the ERH Protein.
  JOURNAL   Mol Cell. 2020 Apr 16;78(2):289-302.e6. doi: 
            10.1016/j.molcel.2020.01.026.
  PUBMED    32302541
REFERENCE   2  (bases 1 to 11590)
  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"; doi: 
            "10.1016/j.molcel.2020.01"; journalName: "Mol Cell"; date: 
            "2020-04-16- 16"; volume: "78"; issue: "2"; pages: "289-302.e6"
COMMENT     Sequence Label: pLentiCRISPRv1.mir451_KO
FEATURES             Location/Qualifiers
     source          1..11590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             295..342
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             358..414
                     /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             415..1014
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(415..434)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     911..931
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1030..1618
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1083..1103)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1501..1512)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1690..1923
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2001..2122
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2038..2057)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2092..2111
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2162..2297
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2317..2336)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2318..2336)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2346..2363)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2346..2362)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2355..2377)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2504..2959
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2591..2610)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2801..2822
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2985..3089
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3090..3158,3159..3950)
                     /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     complement(3308..3327)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4121..4709
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4610..4629
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4863..4880
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4997..5018
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5033..5038,5039..5056,5057..5063)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5071..5087
                     /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)."
     primer_bind     5076..5098
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5095..5111
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5095..5111
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5130..5150
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5132..5150
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5178..5404
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5405..5585
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5632..5757
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6250..6483
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6668..6712
                     /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"
     promoter        6890..7138
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6890..6910
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7061..7080
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7160..7235
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    7283..7400
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7469..7680
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7700..7709
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7709..7732
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7739..7759
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(7784..11590,1..294)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(7784..11590,1..294)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
       61 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
      121 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
      181 atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag
      241 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaagcgt
      301 cctgctgcta ctaagaaagc tggtcaagct aagaaaaaga aagctagcgg cagcggcgcc
      361 accaacttca gcctgctgaa gcaggccggc gacgtggagg agaaccccgg ccccatgacc
      421 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
      481 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
      541 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
      601 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
      661 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
      721 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
      781 tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc
      841 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
      901 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
      961 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaacgcgt
     1021 taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     1081 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     1141 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     1201 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     1261 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     1321 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     1381 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     1441 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     1501 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     1561 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgt
     1621 cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     1681 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta
     1741 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     1801 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     1861 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     1921 gcagtacgta tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     1981 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     2041 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     2101 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     2161 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     2221 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     2281 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     2341 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     2401 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     2461 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     2521 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2581 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2641 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2701 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2761 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2821 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2881 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2941 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3001 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3061 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3121 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3181 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3241 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3301 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3361 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3421 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3481 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3541 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3601 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3661 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3721 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3781 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3841 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3901 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3961 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4021 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4081 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4141 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4201 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4261 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4321 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4381 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4441 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4501 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4561 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4621 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4681 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4741 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4801 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4861 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     4921 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     4981 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     5041 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     5101 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
     5161 tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac
     5221 gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga
     5281 agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg
     5341 acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat
     5401 aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     5461 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     5521 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     5581 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
     5641 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
     5701 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
     5761 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
     5821 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
     5881 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
     5941 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
     6001 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
     6061 gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg
     6121 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
     6181 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
     6241 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
     6301 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
     6361 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
     6421 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
     6481 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
     6541 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
     6601 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
     6661 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
     6721 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
     6781 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
     6841 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt
     6901 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
     6961 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     7021 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     7081 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
     7141 ctgagtttag taatggtaag ttttagagct agaaatagca agttaaaata aggctagtcc
     7201 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgaattctag atcttgagac
     7261 aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag
     7321 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
     7381 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc
     7441 ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     7501 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     7561 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     7621 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     7681 gtgtcgtgac gcgggatccg ccaccatgga ttacaaagac gatgacgata agatggcccc
     7741 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
     7801 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     7861 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     7921 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     7981 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     8041 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     8101 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     8161 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     8221 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     8281 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     8341 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     8401 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     8461 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     8521 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     8581 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     8641 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     8701 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     8761 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     8821 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     8881 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     8941 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     9001 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     9061 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     9121 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     9181 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     9241 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     9301 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     9361 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     9421 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     9481 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     9541 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     9601 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     9661 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     9721 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     9781 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     9841 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     9901 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     9961 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
    10021 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
    10081 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
    10141 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
    10201 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
    10261 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
    10321 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
    10381 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
    10441 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
    10501 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
    10561 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
    10621 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
    10681 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
    10741 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
    10801 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
    10861 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
    10921 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
    10981 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
    11041 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
    11101 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
    11161 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
    11221 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
    11281 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
    11341 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
    11401 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
    11461 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
    11521 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
    11581 cgagatcatc
//
