LOCUS       sequence-362641-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets JAK3 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11691)
  AUTHORS   Hu M, Lei XY, Larson JD, McAlonis M, Ford K, McDonald D, Mach K, 
            Rusert JM, Wechsler-Reya RJ, Mali P
  TITLE     Integrated genome and tissue engineering enables screening of cancer
            vulnerabilities in physiologically relevant perfusable ex vivo 
            cultures.
  JOURNAL   Biomaterials. 2022 Jan;280:121276. doi: 
            10.1016/j.biomaterials.2021.121276. Epub 2021 Dec 2.
  PUBMED    34890975
REFERENCE   2  (bases 1 to 11691)
  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: 
            "Biomaterials."; date: "2022-01"; volume: "280:121276. doi"; pages: 
            " 10.1016/j.biomaterials.2021.121276. Epub 2021 Dec 2"
COMMENT     Sequence Label: CROP_C9_Puro_JAK3
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..34
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        16..34
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        62..288
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             289..469
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    516..641
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1134..1367
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1552..1596
                     /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    1777..1894
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2083..2294
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2313..2322
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2319..6422
                     /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             2319..6422
                     /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             6423..6470
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6471..6494
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6504..6560
                     /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             6561..7160
                     /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(6561..6580)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7057..7077
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7176..7764
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7229..7249)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7647..7658)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7920..8168
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7920..7940
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8091..8110
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8190..8265
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8318..8498
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8576..8697
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8613..8632)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8667..8686
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8737..8872
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8892..8911)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8893..8911)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8921..8938)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8921..8937)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8930..8952)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9079..9534
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9166..9185)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9376..9397
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9560..9664
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9665..9733,9734..10525)
                     /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(9883..9902)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10696..11284
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11185..11204
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11438..11455
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11572..11593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11608..11613,11614..11631,11632..11638)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11646..11662
                     /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     11651..11673
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11670..11686
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11670..11686
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
       61 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      121 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      181 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      241 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      301 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      361 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      421 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      481 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      541 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      601 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      661 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      721 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
      781 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
      841 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
      901 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
      961 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1021 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1081 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1141 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1201 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1261 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1321 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1381 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1441 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1501 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1561 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1621 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1681 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1741 cagaatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg
     1801 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga
     1861 attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga
     1921 tccactttgg cgccggctcg agggggcccg ggtgcaaaga tggataaagt tttaaacaga
     1981 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgt
     2041 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc
     2101 ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt
     2161 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     2221 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     2281 ccgccagaac acaggaccgg ttctagagcg ctgccaccat ggacaagaag tacagcatcg
     2341 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     2401 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     2461 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     2521 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     2581 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     2641 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     2701 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     2761 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     2821 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     2881 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     2941 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     3001 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     3061 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     3121 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     3181 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     3241 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     3301 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     3361 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     3421 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     3481 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     3541 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     3601 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     3661 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     3721 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     3781 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     3841 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     3901 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     3961 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     4021 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     4081 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     4141 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     4201 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     4261 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     4321 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     4381 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     4441 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     4501 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     4561 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     4621 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     4681 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     4741 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     4801 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     4861 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     4921 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     4981 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     5041 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     5101 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     5161 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     5221 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     5281 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     5341 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     5401 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     5461 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     5521 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     5581 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     5641 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     5701 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     5761 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     5821 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     5881 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     5941 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     6001 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     6061 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     6121 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     6181 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     6241 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     6301 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     6361 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     6421 acaagcgacc tgccgccaca aagaaggctg gacaggctaa gaagaagaaa gattacaaag
     6481 acgatgacga taagggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg
     6541 tcgaagagaa tcctggaccg atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     6601 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     6661 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     6721 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgccgtgg
     6781 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     6841 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     6901 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc
     6961 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     7021 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     7081 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     7141 cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat tacaaaattt
     7201 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     7261 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     7321 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     7381 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     7441 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     7501 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     7561 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     7621 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     7681 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     7741 tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt
     7801 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg
     7861 aagacaagat ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag
     7921 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     7981 taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     8041 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     8101 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     8161 gaaacaccgg gaagctgtca aagtcctggg ttttagagct agaaatagca agttaaaata
     8221 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt taagcttggc
     8281 gtaactagat cttgagacac tgctttttgc ttgtactggg tctctctggt tagaccagat
     8341 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     8401 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     8461 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca
     8521 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     8581 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     8641 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     8701 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     8761 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     8821 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg
     8881 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     8941 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     9001 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     9061 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     9121 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     9181 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     9241 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     9301 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     9361 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     9421 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     9481 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     9541 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     9601 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     9661 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     9721 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     9781 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     9841 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     9901 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     9961 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
    10021 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
    10081 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
    10141 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
    10201 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
    10261 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
    10321 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
    10381 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
    10441 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
    10501 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
    10561 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
    10621 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
    10681 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
    10741 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
    10801 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
    10861 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
    10921 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
    10981 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
    11041 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
    11101 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
    11161 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
    11221 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
    11281 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
    11341 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
    11401 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
    11461 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
    11521 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
    11581 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
    11641 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg a
//
