LOCUS       sequence-362605-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets ALK 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_ALK
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    54..75
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(90..95,96..113,114..120)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    128..144
                     /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     133..155
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     152..168
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     152..168
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     187..207
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        189..207
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        235..461
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             462..642
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    689..814
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1307..1540
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1725..1769
                     /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    1950..2067
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2256..2467
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2486..2495
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2492..6595
                     /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             2492..6595
                     /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             6596..6643
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6644..6667
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6677..6733
                     /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             6734..7333
                     /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(6734..6753)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7230..7250
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7349..7937
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7402..7422)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7820..7831)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        8093..8341
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8093..8113
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8264..8283
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8363..8438
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8491..8671
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8749..8870
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8786..8805)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8840..8859
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8910..9045
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9065..9084)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9066..9084)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9094..9111)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9094..9110)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9103..9125)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9252..9707
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9339..9358)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9549..9570
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9733..9837
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9838..9906,9907..10698)
                     /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(10056..10075)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10869..11457
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11358..11377
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11611..11628
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
       61 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
      121 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
      181 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
      241 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
      301 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
      361 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
      421 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
      481 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      541 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      601 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      661 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      721 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      781 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      841 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
      901 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
      961 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1021 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1081 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1141 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     1201 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1261 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     1321 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     1381 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     1441 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     1501 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     1561 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     1621 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1681 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1741 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1801 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1861 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaatc
     1921 ttgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac
     1981 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     2041 aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt
     2101 tggcgccggc tcgagggggc ccgggtgcaa agatggataa agttttaaac agagaggaat
     2161 ctttgcagct aatggacctt ctaggtcttg aaaggagtgg gaattggctc cgtaggtctt
     2221 gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
     2281 ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg
     2341 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
     2401 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
     2461 aacacaggac cggttctaga gcgctgccac catggacaag aagtacagca tcggcctgga
     2521 catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa
     2581 gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc
     2641 cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag
     2701 aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat
     2761 ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga
     2821 taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga
     2881 gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga
     2941 cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat
     3001 cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca
     3061 gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc
     3121 catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc
     3181 cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc
     3241 caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac
     3301 ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt
     3361 tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac
     3421 cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca
     3481 ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat
     3541 tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga
     3601 agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct
     3661 cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat
     3721 cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta
     3781 cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta
     3841 ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga
     3901 ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag
     3961 cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa
     4021 gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt
     4081 gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga
     4141 cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa
     4201 gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc
     4261 cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga
     4321 ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga
     4381 gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca
     4441 gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat
     4501 ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa
     4561 cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa
     4621 agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag
     4681 ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt
     4741 gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac
     4801 ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga
     4861 gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa
     4921 gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat
     4981 caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga
     5041 ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt
     5101 gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa
     5161 gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga
     5221 actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca
     5281 cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat
     5341 ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt
     5401 ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa
     5461 cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta
     5521 cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg
     5581 caaggctacc gccaagtact tcttctacag caacatcatg aactttttca agaccgagat
     5641 taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg
     5701 ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc
     5761 ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat
     5821 cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa
     5881 gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga
     5941 aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga
     6001 aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt
     6061 gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg
     6121 gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc
     6181 caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga
     6241 ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat
     6301 cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt
     6361 gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat
     6421 ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac
     6481 catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca
     6541 gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaagcg
     6601 acctgccgcc acaaagaagg ctggacaggc taagaagaag aaagattaca aagacgatga
     6661 cgataaggga tccggcgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga
     6721 gaatcctgga ccgatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     6781 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     6841 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     6901 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgccg tggcggtctg
     6961 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     7021 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     7081 ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg
     7141 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     7201 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     7261 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     7321 gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag
     7381 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     7441 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     7501 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     7561 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     7621 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     7681 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     7741 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     7801 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     7861 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     7921 ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt
     7981 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     8041 gatttcacca ttatcgtttc agacccacct cccaaccccg aggggaccca gagagggcct
     8101 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattag
     8161 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     8221 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     8281 cgtaacttga aagtatttcg atttcttggc tatatatatc ttgtggaaag gacgaaacac
     8341 cggatgcccg agaagaaggc gtgttttaga gctagaaata gcaagttaaa ataaggctag
     8401 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttaagctt ggcgtaacta
     8461 gatcttgaga cactgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     8521 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     8581 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     8641 cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt
     8701 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     8761 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     8821 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     8881 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     8941 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     9001 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
     9061 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     9121 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     9181 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     9241 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     9301 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     9361 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     9421 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     9481 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     9541 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     9601 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     9661 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     9721 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     9781 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     9841 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     9901 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     9961 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
    10021 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
    10081 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
    10141 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
    10201 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
    10261 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
    10321 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
    10381 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
    10441 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
    10501 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
    10561 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
    10621 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
    10681 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
    10741 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
    10801 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
    10861 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
    10921 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
    10981 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
    11041 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
    11101 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
    11161 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
    11221 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
    11281 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
    11341 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
    11401 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
    11461 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
    11521 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
    11581 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
    11641 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg c
//
