LOCUS       sequence-367165-       11690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets BRAF gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11690)
  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 11690)
  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_BRAF
FEATURES             Location/Qualifiers
     source          1..11690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..45
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        27..45
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        73..299
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             300..480
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    527..652
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1145..1378
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1563..1607
                     /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    1788..1905
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2094..2305
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2324..2333
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2330..6433
                     /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             2330..6433
                     /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             6434..6481
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6482..6505
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6515..6571
                     /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             6572..7171
                     /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(6572..6591)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7068..7088
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7187..7775
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7240..7260)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7658..7669)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7931..8179
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7931..7951
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8102..8121
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8201..8275
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8328..8508
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8586..8707
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8623..8642)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8677..8696
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8747..8882
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8902..8921)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8903..8921)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8931..8948)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8931..8947)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8940..8962)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9089..9544
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9176..9195)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9386..9407
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9570..9674
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9675..9743,9744..10535)
                     /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(9893..9912)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10706..11294
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11195..11214
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11448..11465
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11582..11603
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11618..11623,11624..11641,11642..11648)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11656..11672
                     /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     11661..11683
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(11680..11690,1..6)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(11680..11690,1..6)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
       61 ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga
      121 gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa
      181 ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa
      241 ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg
      301 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
      361 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
      421 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
      481 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
      541 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
      601 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
      661 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
      721 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
      781 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
      841 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
      901 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
      961 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
     1021 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     1081 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     1141 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     1201 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     1261 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     1321 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     1381 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     1441 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     1501 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     1561 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     1621 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     1681 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     1741 cattatcgtt tcagaatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa
     1801 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     1861 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     1921 gacagcagag atccactttg gcgccggctc gagggggccc gggtgcaaag atggataaag
     1981 ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa aggagtggga
     2041 attggctccg taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga
     2101 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc
     2161 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     2221 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     2281 caacgggttt gccgccagaa cacaggaccg gttctagagc gctgccacca tggacaagaa
     2341 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     2401 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     2461 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     2521 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     2581 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     2641 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     2701 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     2761 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     2821 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     2881 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     2941 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     3001 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     3061 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     3121 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     3181 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     3241 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     3301 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     3361 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     3421 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     3481 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     3541 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     3601 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     3661 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     3721 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     3781 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     3841 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     3901 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     3961 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     4021 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     4081 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     4141 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     4201 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     4261 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     4321 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     4381 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     4441 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     4501 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     4561 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     4621 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     4681 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     4741 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     4801 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     4861 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     4921 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     4981 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     5041 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     5101 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     5161 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     5221 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     5281 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     5341 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     5401 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     5461 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     5521 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     5581 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     5641 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     5701 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     5761 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     5821 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     5881 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     5941 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     6001 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     6061 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     6121 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     6181 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     6241 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     6301 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     6361 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     6421 gctgggaggc gacaagcgac ctgccgccac aaagaaggct ggacaggcta agaagaagaa
     6481 agattacaaa gacgatgacg ataagggatc cggcgcaaca aacttctctc tgctgaaaca
     6541 agccggagat gtcgaagaga atcctggacc gatgaccgag tacaagccca cggtgcgcct
     6601 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     6661 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     6721 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     6781 cgccgccgtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     6841 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     6901 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt
     6961 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     7021 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     7081 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     7141 ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga
     7201 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     7261 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     7321 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     7381 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     7441 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     7501 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     7561 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     7621 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     7681 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     7741 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
     7801 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     7861 cactcccaac gaagacaaga tttcaccatt atcgtttcag acccacctcc caaccccgag
     7921 gggacccaga gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     7981 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     8041 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     8101 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     8161 gtggaaagga cgaaacaccg gggccaggct ctgttcaacg gttttagagc tagaaatagc
     8221 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     8281 taagcttggc gtaactagat cttgagacac tgctttttgc ttgtactggg tctctctggt
     8341 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     8401 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     8461 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta
     8521 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     8581 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     8641 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     8701 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     8761 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     8821 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     8881 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     8941 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     9001 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     9061 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     9121 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     9181 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     9241 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     9301 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     9361 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     9421 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     9481 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     9541 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     9601 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     9661 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     9721 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     9781 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     9841 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     9901 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     9961 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
    10021 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
    10081 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
    10141 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
    10201 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
    10261 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
    10321 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
    10381 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
    10441 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
    10501 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
    10561 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
    10621 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
    10681 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
    10741 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
    10801 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
    10861 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
    10921 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
    10981 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
    11041 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
    11101 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
    11161 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
    11221 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
    11281 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
    11341 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
    11401 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
    11461 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
    11521 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
    11581 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
    11641 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
//
