LOCUS       sequence-468886-       11883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CRISPRoff-mScarletI 
            (DNMT3A-DNMT3L-XTEN80-dCas9-HA-2xNLS-mScarletI-KRAB) downstream of 
            the CAG promoter for gene epigenetic silencing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11883)
  AUTHORS   Yin JA, Frick L, Scheidmann MC, Liu T, Trevisan C, Dhingra A, 
            Spinelli A, Wu Y, Yao L, Vena DL, Knapp B, Guo J, De Cecco E, Ging 
            K, Armani A, Oakeley EJ, Nigsch F, Jenzer J, Haegele J, Pikusa M, 
            Tager J, Rodriguez-Nieto S, Bouris V, Ribeiro R, Baroni F, Bedi MS, 
            Berry S, Losa M, Hornemann S, Kampmann M, Pelkmans L, Hoepfner D, 
            Heutink P, Aguzzi A
  TITLE     Arrayed CRISPR libraries for the genome-wide activation, deletion 
            and silencing of human protein-coding genes.
  JOURNAL   Nat Biomed Eng. 2024 Dec 4. doi: 10.1038/s41551-024-01278-4.
  PUBMED    39633028
REFERENCE   2  (bases 1 to 11883)
  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: "Nat Biomed 
            Eng. 2024 Dec 4. doi: 10.1038/s41551-024-01278-4."
COMMENT     Sequence Label: CRISPRoff-mScarletI
FEATURES             Location/Qualifiers
     source          1..11883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..659
                     /label=chicken beta-actin promoter
     intron          660..1676
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1600..1622
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1684..1703
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(3647..3673,3674..3676,3677..6163,6164..6166,
                     6167..7750)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7754..7780
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7760..7786
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             7799..7819
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7826..7846
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7886..8581
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(8019..8037)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     8287..8306
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             8627..8812
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     primer_bind     complement(8904..8923)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    8969..9024
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(9023..9042)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(9385..9401)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9385..9401)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9398..9420)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9409..9425
                     /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(9433..9439,9440..9457,9458..9463))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9478..9499
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        9558..9753
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      9604..9739
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    9759..9893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9796..9815)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9850..9869
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9961..9978)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10132..10720)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10212..10231)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10891..11682,11683..11751))
                     /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     11514..11533
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11752..11856)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg
      541 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
      601 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
      661 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
      721 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
      781 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
      841 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct
      961 gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg
     1021 gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     1081 gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc
     1141 cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg
     1201 gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg
     1261 ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg
     1321 gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc
     1381 agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc
     1441 cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg
     1501 gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc
     1561 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
     1621 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
     1681 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc tgcagtcgac
     1741 ggtaccgcgg gcccgggatc caccggtcgc caccatgaac catgaccagg aatttgaccc
     1801 cccaaaggtt tacccacctg tgccagctga gaagaggaag cccatccgcg tgctgtctct
     1861 ctttgatggg attgctacag ggctcctggt gctgaaggac ctgggcatcc aagtggaccg
     1921 ctacattgcc tccgaggtgt gtgaggactc catcacggtg ggcatggtgc ggcaccaggg
     1981 aaagatcatg tacgtcgggg acgtccgcag cgtcacacag aagcatatcc aggagtgggg
     2041 cccattcgac ctggtgattg gaggcagtcc ctgcaatgac ctctccattg tcaaccctgc
     2101 ccgcaaggga ctttatgagg gtactggccg cctcttcttt gagttctacc gcctcctgca
     2161 tgatgcgcgg cccaaggagg gagatgatcg ccccttcttc tggctctttg agaatgtggt
     2221 ggccatgggc gttagtgaca agagggacat ctcgcgattt cttgagtcta accccgtgat
     2281 gattgacgcc aaagaagtgt ctgctgcaca cagggcccgt tacttctggg gtaaccttcc
     2341 tggcatgaac aggcctttgg catccactgt gaatgataag ctggagctgc aagagtgtct
     2401 ggagcacggc agaatagcca agttcagcaa agtgaggacc attaccacca ggtcaaactc
     2461 tataaagcag ggcaaagacc agcatttccc cgtcttcatg aacgagaagg aggacatcct
     2521 gtggtgcact gaaatggaaa gggtgtttgg cttccccgtc cactacacag acgtctccaa
     2581 catgagccgc ttggcgaggc agagactgct gggccgatcg tggagcgtgc cggtcatccg
     2641 ccacctcttc gctccgctga aggaatattt tgcttgtgtg tctagcggca atagtaacgc
     2701 taacagccgc gggccgagct tcagcagcgg cctggtgccg ttaagcttgc gcggcagcca
     2761 tatgggccct atggagatat acaagacagt gtctgcatgg aagagacagc cagtgcgggt
     2821 actgagcctc ttcagaaaca tcgacaaggt actaaagagt ttgggcttct tggaaagcgg
     2881 ttctggttct gggggaggaa cgctgaagta cgtggaagat gtcacaaatg tcgtgaggag
     2941 agacgtggag aaatggggcc cctttgacct ggtgtacggc tcgacgcagc ccctaggcag
     3001 ctcttgtgat cgctgtcccg gctggtacat gttccagttc caccggatcc tgcagtatgc
     3061 gctgcctcgc caggagagtc agcggccctt cttctggata ttcatggaca atctgctgct
     3121 gactgaggat gaccaagaga caactacccg cttccttcag acagaggctg tgaccctcca
     3181 ggatgtccgt ggcagagact accagaatgc tatgcgggtg tggagcaaca ttccagggct
     3241 gaagagcaag catgcgcccc tgaccccaaa ggaagaagag tatctgcaag cccaagtcag
     3301 aagcaggagc aagctggacg ccccgaaagt tgacctcctg gtgaagaact gccttctccc
     3361 gctgagagag tacttcaagt atttttctca aaactcactt cctcttggag ggccgagctc
     3421 tggcgcaccc ccaccaagtg gagggtctcc tgccgggtcc ccaacatcta ctgaagaagg
     3481 caccagcgaa tccgcaacgc ccgagtcagg ccctggtacc tccacagaac catctgaagg
     3541 tagtgcgcct ggttccccag ctggaagccc tacttccacc gaagaaggca cgtcaaccga
     3601 accaagtgaa ggatctgccc ctgggaccag cactgaacca tctgagatgg acaagaagta
     3661 ttctatcgga ctggccatcg ggactaatag cgtcgggtgg gccgtgatca ctgacgagta
     3721 caaggtgccc tctaagaagt tcaaggtgct cgggaacacc gaccggcatt ccatcaagaa
     3781 aaatctgatc ggagctctcc tctttgattc aggggagacc gctgaagcaa cccgcctcaa
     3841 gcggactgct agacggcggt acaccaggag gaagaaccgg atttgttacc ttcaagagat
     3901 attctccaac gaaatggcaa aggtcgacga cagcttcttc cataggctgg aagaatcatt
     3961 cctcgtggaa gaggataaga agcatgaacg gcatcccatc ttcggtaata tcgtcgacga
     4021 ggtggcctat cacgagaaat acccaaccat ctaccatctt cgcaaaaagc tggtggactc
     4081 aaccgacaag gcagacctcc ggcttatcta cctggccctg gcccacatga tcaagttcag
     4141 aggccacttc ctgatcgagg gcgacctcaa tcctgacaat agcgatgtgg ataaactgtt
     4201 catccagctg gtgcagactt acaaccagct ctttgaagag aaccccatca atgcaagcgg
     4261 agtcgatgcc aaggccattc tgtcagcccg gctgtcaaag agccgcagac ttgagaatct
     4321 tatcgctcag ctgccgggtg aaaagaaaaa tggactgttc gggaacctga ttgctctttc
     4381 acttgggctg actcccaatt tcaagtctaa tttcgacctg gcagaggatg ccaagctgca
     4441 actgtccaag gacacctatg atgacgatct cgacaacctc ctggcccaga tcggtgacca
     4501 atacgccgac cttttccttg ctgctaagaa tctttctgac gccatcctgc tgtctgacat
     4561 tctccgcgtg aacactgaaa tcaccaaggc ccctctttca gcttcaatga ttaagcggta
     4621 tgatgagcac caccaggacc tgaccctgct taaggcactc gtccggcagc agcttccgga
     4681 gaagtacaag gaaatcttct ttgaccagtc aaagaatgga tacgccggct acatcgacgg
     4741 aggtgcctcc caagaggaat tttataagtt tatcaaacct atccttgaga agatggacgg
     4801 caccgaagag ctcctcgtga aactgaatcg ggaggatctg ctgcggaagc agcgcacttt
     4861 cgacaatggg agcattcccc accagatcca tcttggggag cttcacgcca tccttcggcg
     4921 ccaagaggac ttctacccct ttcttaagga caacagggag aagattgaga aaattctcac
     4981 tttccgcatc ccctactacg tgggacccct cgccagagga aatagccggt ttgcttggat
     5041 gaccagaaag tcagaagaaa ctatcactcc ctggaacttc gaagaggtgg tggacaaggg
     5101 agccagcgct cagtcattca tcgaacggat gactaacttc gataagaacc tccccaatga
     5161 gaaggtcctg ccgaaacatt ccctgctcta cgagtacttt accgtgtaca acgagctgac
     5221 caaggtgaaa tatgtcaccg aagggatgag gaagcccgca ttcctgtcag gcgaacaaaa
     5281 gaaggcaatt gtggaccttc tgttcaagac caatagaaag gtgaccgtga agcagctgaa
     5341 ggaggactat ttcaagaaaa ttgaatgctt cgactctgtg gagattagcg gggtcgaaga
     5401 tcggttcaac gcaagcctgg gtacctacca tgatctgctt aagatcatca aggacaagga
     5461 ttttctggac aatgaggaga acgaggacat ccttgaggac attgtcctga ctctcactct
     5521 gttcgaggac cgggaaatga tcgaggagag gcttaagacc tacgcccatc tgttcgacga
     5581 taaagtgatg aagcaactta aacggagaag atataccgga tggggacgcc ttagccgcaa
     5641 actcatcaac ggaatccggg acaaacagag cggaaagacc attcttgatt tccttaagag
     5701 cgacggattc gctaatcgca acttcatgca acttatccat gatgattccc tgacctttaa
     5761 ggaggacatc cagaaggccc aagtgtctgg acaaggtgac tcactgcacg agcatatcgc
     5821 aaatctggct ggttcacccg ctattaagaa gggtattctc cagaccgtga aagtcgtgga
     5881 cgagctggtc aaggtgatgg gtcgccataa accagagaac attgtcatcg agatggccag
     5941 ggaaaaccag actacccaga agggacagaa gaacagcagg gagcggatga aaagaattga
     6001 ggaagggatt aaggagctcg ggtcacagat ccttaaagag cacccggtgg aaaacaccca
     6061 gcttcagaat gagaagctct atctgtacta ccttcaaaat ggacgcgata tgtatgtgga
     6121 ccaagagctt gatatcaaca ggctctcaga ctacgacgtg gacgccatcg tccctcagag
     6181 cttcctcaaa gacgactcaa ttgacaataa ggtgctgact cgctcagaca agaaccgggg
     6241 aaagtcagat aacgtgccct cagaggaagt cgtgaaaaag atgaagaact attggcgcca
     6301 gcttctgaac gcaaagctga tcactcagcg gaagttcgac aatctcacta aggctgagag
     6361 gggcggactg agcgaactgg acaaagcagg attcattaaa cggcaacttg tggagactcg
     6421 gcagattact aaacatgtcg cccaaatcct tgactcacgc atgaatacca agtacgacga
     6481 aaacgacaaa cttatccgcg aggtgaaggt gattaccctg aagtccaagc tggtcagcga
     6541 tttcagaaag gactttcaat tctacaaagt gcgggagatc aataactatc atcatgctca
     6601 tgacgcatat ctgaatgccg tggtgggaac cgccctgatc aagaagtacc caaagctgga
     6661 aagcgagttc gtgtacggag actacaaggt ctacgacgtg cgcaagatga ttgccaaatc
     6721 tgagcaggag atcggaaagg ccaccgcaaa gtacttcttc tacagcaaca tcatgaattt
     6781 cttcaagacc gaaatcaccc ttgcaaacgg tgagatccgg aagaggccgc tcatcgagac
     6841 taatggggag actggcgaaa tcgtgtggga caagggcaga gatttcgcta ccgtgcgcaa
     6901 agtgctttct atgcctcaag tgaacatcgt gaagaaaacc gaggtgcaaa ccggaggctt
     6961 ttctaaggaa tcaatcctcc ccaagcgcaa ctccgacaag ctcattgcaa ggaagaagga
     7021 ttgggaccct aagaagtacg gcggattcga ttcaccaact gtggcttatt ctgtcctggt
     7081 cgtggctaag gtggaaaaag gaaagtctaa gaagctcaag agcgtgaagg aactgctggg
     7141 tatcaccatt atggagcgca gctccttcga gaagaaccca attgactttc tcgaagccaa
     7201 aggttacaag gaagtcaaga aggaccttat catcaagctc ccaaagtata gcctgttcga
     7261 actggagaat gggcggaagc ggatgctcgc ctccgctggc gaacttcaga agggtaatga
     7321 gctggctctc ccctccaagt acgtgaattt cctctacctt gcaagccatt acgagaagct
     7381 gaaggggagc cccgaggaca acgagcaaaa gcaactgttt gtggagcagc ataagcatta
     7441 tctggacgag atcattgagc agatttccga gttttctaaa cgcgtcattc tcgctgatgc
     7501 caacctcgat aaagtcctta gcgcatacaa taagcacaga gacaaaccaa ttcgggagca
     7561 ggctgagaat atcatccacc tgttcaccct caccaatctt ggtgcccctg ccgcattcaa
     7621 gtacttcgac accaccatcg accggaaacg ctatacctcc accaaagaag tgctggacgc
     7681 caccctcatc caccagagca tcaccggact ttacgaaact cggattgacc tctcacagct
     7741 cggaggggat gcctatccct atgacgtgcc cgattatgcc agcctgggca gcggctcccc
     7801 caagaaaaaa cgcaaggtgg aagatcctaa gaaaaagcgg aaagtggacg gcattggtag
     7861 tgggagcaac ggcagcagcg gatccatggt gagcaagggc gaggcagtga tcaaggagtt
     7921 catgcggttc aaggtgcaca tggagggctc catgaacggc cacgagttcg agatcgaggg
     7981 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     8041 tggccccctg cccttctcct gggacatcct gtcccctcag ttcatgtacg gctccagggc
     8101 cttcatcaag caccccgccg acatccccga ctactataag cagtccttcc ccgagggctt
     8161 caagtgggag cgcgtgatga acttcgagga cggcggcgcc gtgaccgtga cccaggacac
     8221 ctccctggag gacggcaccc tgatctacaa ggtgaagctc cgcggcacca acttccctcc
     8281 tgacggcccc gtaatgcaga agaagacaat gggctgggaa gcgtccaccg agcggttgta
     8341 ccccgaggac ggcgtgctga agggcgacat taagatggcc ctgcgcctga aggacggcgg
     8401 ccgctacctg gcggacttca agaccaccta caaggccaag aagcccgtgc agatgcccgg
     8461 cgcctacaac gtcgaccgca agttggacat cacctcccac aacgaggact acaccgtggt
     8521 ggaacagtac gaacgctccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     8581 gggcggtggc ggagggatgg atgctaagtc actaactgcc tggtcccgga cactggtgac
     8641 cttcaaggat gtatttgtgg acttcaccag ggaggagtgg aagctgctgg acactgctca
     8701 gcagatcgtg tacagaaatg tgatgctgga gaactataag aacctggttt ccttgggtta
     8761 tcagcttact aagccagatg tgatcctccg gttggagaag ggagaagagc cctagtaagc
     8821 ggccgcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     8881 ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg
     8941 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     9001 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa
     9061 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg
     9121 aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc
     9181 ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta
     9241 tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc
     9301 ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg
     9361 cagcccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     9421 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     9481 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     9541 gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     9601 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     9661 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     9721 ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata
     9781 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     9841 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atccgctgca
     9901 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     9961 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
    10021 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
    10081 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
    10141 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
    10201 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
    10261 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
    10321 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
    10381 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
    10441 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
    10501 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
    10561 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
    10621 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
    10681 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
    10741 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
    10801 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
    10861 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
    10921 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
    10981 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
    11041 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
    11101 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
    11161 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
    11221 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
    11281 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
    11341 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
    11401 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
    11461 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
    11521 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
    11581 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
    11641 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
    11701 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
    11761 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
    11821 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
    11881 tgg
//
