LOCUS       sequence-362667-       11690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets RET 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_RET
FEATURES             Location/Qualifiers
     source          1..11690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..47
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        29..47
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        75..301
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             302..482
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    529..654
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1147..1380
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1565..1609
                     /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    1790..1907
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2096..2307
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2326..2335
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2332..6435
                     /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             2332..6435
                     /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             6436..6483
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6484..6507
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6517..6573
                     /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             6574..7173
                     /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(6574..6593)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7070..7090
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7189..7777
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7242..7262)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7660..7671)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7933..8173
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     7933..7953
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8104..8123
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8202..8277
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8330..8510
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8588..8709
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8625..8644)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8679..8698
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8749..8884
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8904..8923)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8905..8923)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8933..8950)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8933..8949)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8942..8964)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9091..9546
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9178..9197)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9388..9409
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9572..9676
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9677..9745,9746..10537)
                     /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(9895..9914)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10708..11296
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11197..11216
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11450..11467
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11584..11605
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11620..11625,11626..11643,11644..11650)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11658..11674
                     /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     11663..11685
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(11682..11690,1..8)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(11682..11690,1..8)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
       61 agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat
      121 gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt
      181 aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg
      241 aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa
      301 cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
      361 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
      421 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
      481 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
      541 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
      601 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
      661 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
      721 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
      781 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
      841 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
      901 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
      961 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     1021 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     1081 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     1141 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     1201 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     1261 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     1321 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     1381 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     1441 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     1501 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     1561 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     1621 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     1681 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     1741 accattatcg tttcagaatc ttgagacaaa tggcagtatt catccacaat tttaaaagaa
     1801 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
     1861 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
     1921 gggacagcag agatccactt tggcgccggc tcgagggggc ccgggtgcaa agatggataa
     1981 agttttaaac agagaggaat ctttgcagct aatggacctt ctaggtcttg aaaggagtgg
     2041 gaattggctc cgtaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca
     2101 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt
     2161 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
     2221 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
     2281 cgcaacgggt ttgccgccag aacacaggac cggttctaga gcgctgccac catggacaag
     2341 aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac
     2401 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc
     2461 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg
     2521 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa
     2581 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag
     2641 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg
     2701 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg
     2761 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag
     2821 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag
     2881 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc
     2941 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa
     3001 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc
     3061 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa
     3121 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc
     3181 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc
     3241 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag
     3301 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg
     3361 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt
     3421 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg
     3481 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg
     3541 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg
     3601 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc
     3661 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc
     3721 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac
     3781 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc
     3841 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag
     3901 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag
     3961 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag
     4021 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg
     4081 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
     4141 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
     4201 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
     4261 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
     4321 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
     4381 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
     4441 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
     4501 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
     4561 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
     4621 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
     4681 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac
     4741 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
     4801 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct
     4861 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac
     4921 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
     4981 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc
     5041 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
     5101 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
     5161 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
     5221 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
     5281 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
     5341 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
     5401 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
     5461 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc
     5521 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg
     5581 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc
     5641 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag
     5701 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg
     5761 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg
     5821 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa
     5881 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg
     5941 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga
     6001 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag
     6061 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag
     6121 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc
     6181 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga
     6241 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc
     6301 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg
     6361 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct
     6421 cagctgggag gcgacaagcg acctgccgcc acaaagaagg ctggacaggc taagaagaag
     6481 aaagattaca aagacgatga cgataaggga tccggcgcaa caaacttctc tctgctgaaa
     6541 caagccggag atgtcgaaga gaatcctgga ccgatgaccg agtacaagcc cacggtgcgc
     6601 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     6661 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     6721 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     6781 ggcgccgccg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     6841 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     6901 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     6961 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     7021 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     7081 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     7141 acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg
     7201 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     7261 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     7321 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     7381 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     7441 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     7501 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     7561 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     7621 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     7681 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     7741 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt
     7801 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     7861 ttcactccca acgaagacaa gatttcacca ttatcgtttc agacccacct cccaaccccg
     7921 aggggaccca gagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     7981 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     8041 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     8101 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     8161 ttgtggaaag gacgaacacc gcggtgagca ggggaaagcc ggttttagag ctagaaatag
     8221 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     8281 tttaagcttg gcgtaactag atcttgagac actgcttttt gcttgtactg ggtctctctg
     8341 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     8401 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     8461 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag
     8521 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag
     8581 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     8641 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     8701 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta
     8761 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     8821 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     8881 ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
     8941 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     9001 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     9061 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     9121 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     9181 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     9241 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     9301 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     9361 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     9421 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     9481 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     9541 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     9601 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     9661 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     9721 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     9781 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     9841 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     9901 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     9961 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
    10021 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
    10081 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
    10141 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
    10201 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
    10261 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
    10321 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
    10381 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
    10441 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
    10501 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
    10561 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
    10621 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
    10681 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
    10741 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
    10801 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
    10861 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
    10921 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
    10981 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
    11041 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
    11101 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
    11161 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
    11221 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
    11281 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
    11341 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
    11401 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
    11461 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
    11521 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
    11581 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
    11641 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
//
