LOCUS       sequence-430318-       13680 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9-D10A nickase under a CAG promoter together with a 
            blasticidin resistance gene, along with a U6 promoter driving the 
            sgCTG (target sequence: (CUG)6). Suitable for lentivirus packaging..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13680)
  AUTHORS   Alvaro Murillo, Melanie Alpaugh, Meghan Larin, Emma L. Randall, 
            Laura Heraty, Ruban Rex Durairaj, Alys N. Aston, Alysha S. Taylor, 
            Alex Mas Monteys, Nina Stoberl, Aeverie E. R. Heuchan, Pascale 
            Aeschlimann, Soumyasree Bhattacharyya, Nicholas D. Allen, Jack 
            Puymirat, Beverly L. Davidson, Francesca Cicchetti, Mariah Lelos, 
            Vincent Dion
  TITLE     Cas9 nickase-mediated contraction of CAG/CTG repeats at multiple 
            disease loci
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 13680)
  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: "bioRxiv"
COMMENT     Sequence Label: pLenti-U6-sgCTG-CAG-Cas9D10A nickase-Blast
FEATURES             Location/Qualifiers
     source          1..13680
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2651..2891
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     2651..2671
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2822..2841
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2929..3004
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        3100..3403
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3405..3682
                     /label=chicken beta-actin promoter
     regulatory      3701..3710
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3707..3733,3734..3736,3737..7810)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3707..7810
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7811..7858
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7859..7882
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7892..7948
                     /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             7949..8347
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8372..8960
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8425..8445)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8843..8854)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8963..8979)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8964..8980)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9485..9665
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(9691..9708)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9697..9921
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9967..10395
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10054..10073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10264..10285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10404..10424)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10409..10738
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10589..10724
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10651..10670
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10786..10833
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10852..11226
                     /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    11356..11477
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11393..11412)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11447..11466
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11526..11542)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11526..11542)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11539..11561)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11550..11566
                     /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(11574..11580,11581..11598,11599..11604))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11619..11640
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11757..11774)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11928..12516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12008..12027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12687..13478,13479..13547))
                     /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     13310..13329
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13548..13652)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggctgtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta agctacctag gtcttgaaag gagtgggaat tggctccggt
     2641 gcccgtcagt gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     2701 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     2761 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     2821 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     2881 gtggaaagga cgaattcgat atcaagcttc tgctgctgct gctgctgcgt tttagagcta
     2941 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     3001 gtgctttttt ttctagaggg taccggggcc cgcctaggta gttattaata gtaatcaatt
     3061 acggggtcat tagttcatag cccatatatg gagttccggc gttacataac ttacggtaaa
     3121 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     3181 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     3241 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     3301 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     3361 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
     3421 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
     3481 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccrggsgg
     3541 ggsggggsgg ggsgrggggs ggggcggggc gaggcggaga ggtgcggcgg cagccaatca
     3601 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
     3661 aaagcgaagc gcgcggcggg cggaccggtt ctagagcgct gccaccatgg acaagaagta
     3721 cagcatcggc ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     3781 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     3841 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     3901 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     3961 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     4021 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     4081 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     4141 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     4201 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     4261 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     4321 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     4381 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgag
     4441 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     4501 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     4561 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     4621 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     4681 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     4741 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     4801 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     4861 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     4921 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     4981 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     5041 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     5101 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     5161 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     5221 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     5281 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     5341 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     5401 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     5461 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     5521 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     5581 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     5641 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     5701 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     5761 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     5821 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     5881 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     5941 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     6001 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     6061 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     6121 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     6181 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     6241 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     6301 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     6361 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     6421 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     6481 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     6541 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     6601 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     6661 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     6721 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     6781 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     6841 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     6901 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     6961 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     7021 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     7081 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     7141 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     7201 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     7261 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     7321 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     7381 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     7441 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     7501 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     7561 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     7621 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     7681 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     7741 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     7801 gggaggcgac aagcgacctg ccgccacaaa gaaggctgga caggctaaga agaagaaaga
     7861 ttacaaagac gatgacgata agggatccgg cgcaacaaac ttctctctgc tgaaacaagc
     7921 cggagatgtc gaagagaatc ctggaccgat ggccaagcct ttgtctcaag aagaatccac
     7981 cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt
     8041 cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt
     8101 tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg
     8161 caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg
     8221 acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag
     8281 tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga
     8341 gggctaagaa ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga
     8401 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     8461 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     8521 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     8581 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     8641 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     8701 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     8761 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     8821 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     8881 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     8941 cctttgggcc gcctccccgc atcgataccg tcgacctcga gacctagaaa aacatggagc
     9001 aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga
     9061 ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa
     9121 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     9181 ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc
     9241 tgattggcag aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg
     9301 ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac
     9361 ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga
     9421 gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg
     9481 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     9541 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     9601 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     9661 tagcagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     9721 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     9781 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     9841 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     9901 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     9961 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
    10021 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
    10081 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
    10141 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
    10201 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
    10261 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
    10321 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
    10381 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
    10441 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
    10501 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
    10561 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
    10621 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
    10681 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
    10741 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
    10801 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag
    10861 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
    10921 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
    10981 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
    11041 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
    11101 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
    11161 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
    11221 gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
    11281 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
    11341 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
    11401 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
    11461 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
    11521 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
    11581 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
    11641 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
    11701 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
    11761 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
    11821 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
    11881 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
    11941 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
    12001 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
    12061 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
    12121 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
    12181 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
    12241 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
    12301 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
    12361 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
    12421 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
    12481 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
    12541 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
    12601 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
    12661 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
    12721 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
    12781 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
    12841 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
    12901 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
    12961 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
    13021 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
    13081 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
    13141 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
    13201 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
    13261 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
    13321 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
    13381 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
    13441 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
    13501 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
    13561 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
    13621 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
//
