LOCUS       sequence-362604-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets ADA 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_ADA
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    32..48
                     /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     37..59
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     56..72
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     56..72
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     91..111
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        93..111
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        139..365
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             366..546
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    593..718
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1211..1444
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1629..1673
                     /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    1854..1971
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2160..2371
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2390..2399
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2396..6499
                     /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             2396..6499
                     /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             6500..6547
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6548..6571
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6581..6637
                     /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             6638..7237
                     /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(6638..6657)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7134..7154
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7253..7841
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7306..7326)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7724..7735)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7997..8245
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7997..8017
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8168..8187
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8267..8342
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8395..8575
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8653..8774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8690..8709)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8744..8763
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8814..8949
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8969..8988)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8970..8988)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8998..9015)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8998..9014)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9007..9029)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9156..9611
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9243..9262)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9453..9474
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9637..9741
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9742..9810,9811..10602)
                     /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(9960..9979)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10773..11361
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11262..11281
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11515..11532
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11649..11670
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11685..11690,11691,1..17,18..24)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
       61 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
      121 ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa
      181 cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg
      241 aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg
      301 gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca
      361 taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
      421 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
      481 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
      541 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg
      601 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag
      661 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag
      721 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa
      781 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt
      841 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc
      901 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta
      961 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga
     1021 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag
     1081 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
     1141 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
     1201 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
     1261 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
     1321 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
     1381 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
     1441 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg
     1501 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg
     1561 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc
     1621 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt
     1681 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct
     1741 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
     1801 attcaccatt atcgtttcag aatcttgaga caaatggcag tattcatcca caattttaaa
     1861 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     1921 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     1981 tacagggaca gcagagatcc actttggcgc cggctcgagg gggcccgggt gcaaagatgg
     2041 ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga
     2101 gtgggaattg gctccgtagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg
     2161 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgatc
     2221 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     2281 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     2341 ttttcgcaac gggtttgccg ccagaacaca ggaccggttc tagagcgctg ccaccatgga
     2401 caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac
     2461 cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag
     2521 catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac
     2581 ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct
     2641 gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga
     2701 agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat
     2761 cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact
     2821 ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat
     2881 caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga
     2941 caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa
     3001 cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct
     3061 ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat
     3121 tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc
     3181 caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat
     3241 cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct
     3301 gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat
     3361 caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca
     3421 gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta
     3481 cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa
     3541 gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca
     3601 gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat
     3661 tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa
     3721 gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt
     3781 cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt
     3841 ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct
     3901 gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa
     3961 cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg
     4021 cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa
     4081 gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg
     4141 cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa
     4201 ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac
     4261 cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct
     4321 gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct
     4381 gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt
     4441 cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct
     4501 gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga
     4561 gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa
     4621 ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga
     4681 aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa
     4741 gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga
     4801 aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat
     4861 gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt
     4921 gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa
     4981 gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta
     5041 ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa
     5101 ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt
     5161 ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa
     5221 gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct
     5281 ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca
     5341 ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc
     5401 taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat
     5461 cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat
     5521 catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct
     5581 gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac
     5641 cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac
     5701 aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag
     5761 aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc
     5821 tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga
     5881 gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct
     5941 ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc
     6001 cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa
     6061 gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta
     6121 tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca
     6181 caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct
     6241 ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat
     6301 cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc
     6361 cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt
     6421 gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct
     6481 gtctcagctg ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa
     6541 gaagaaagat tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct
     6601 gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg accgagtaca agcccacggt
     6661 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     6721 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     6781 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     6841 cgacggcgcc gccgtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     6901 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     6961 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     7021 cggagtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     7081 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     7141 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     7201 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc
     7261 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     7321 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     7381 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     7441 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     7501 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     7561 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     7621 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     7681 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     7741 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     7801 ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg
     7861 acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg
     7921 ctaattcact cccaacgaag acaagatttc accattatcg tttcagaccc acctcccaac
     7981 cccgagggga cccagagagg gcctatttcc catgattcct tcatatttgc atatacgata
     8041 caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca
     8101 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     8161 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     8221 tatcttgtgg aaaggacgaa acaccgatag cctcccggca gcccctgttt tagagctaga
     8281 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     8341 gcttttttaa gcttggcgta actagatctt gagacactgc tttttgcttg tactgggtct
     8401 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     8461 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     8521 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg
     8581 tatagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca
     8641 gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     8701 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     8761 atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc
     8821 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     8881 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     8941 ttggaggcct agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     9001 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     9061 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     9121 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     9181 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     9241 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     9301 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     9361 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     9421 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     9481 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     9541 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     9601 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     9661 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     9721 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     9781 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     9841 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     9901 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     9961 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
    10021 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
    10081 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
    10141 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
    10201 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
    10261 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
    10321 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
    10381 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
    10441 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
    10501 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
    10561 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
    10621 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
    10681 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
    10741 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
    10801 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
    10861 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
    10921 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
    10981 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
    11041 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
    11101 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
    11161 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
    11221 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
    11281 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
    11341 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
    11401 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
    11461 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
    11521 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
    11581 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
    11641 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca c
//
