LOCUS       sequence-362633-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets GART 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_GART
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    22..38
                     /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     27..49
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     46..62
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     46..62
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     81..101
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        83..101
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        129..355
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             356..536
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    583..708
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1201..1434
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1619..1663
                     /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    1844..1961
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2150..2361
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2380..2389
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2386..6489
                     /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             2386..6489
                     /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             6490..6537
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6538..6561
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6571..6627
                     /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             6628..7227
                     /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(6628..6647)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7124..7144
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7243..7831
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7296..7316)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7714..7725)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7987..8235
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7987..8007
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8158..8177
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8257..8332
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8385..8565
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8643..8764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8680..8699)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8734..8753
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8804..8939
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8959..8978)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8960..8978)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8988..9005)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8988..9004)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8997..9019)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9146..9601
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9233..9252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9443..9464
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9627..9731
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9732..9800,9801..10592)
                     /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(9950..9969)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10763..11351
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11252..11271
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11505..11522
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11639..11660
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11675..11680,11681..11691,1..7,8..14)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
       61 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
      121 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
      181 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
      241 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
      301 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
      361 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      421 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      481 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      541 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      601 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
      661 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
      721 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
      781 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
      841 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
      901 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
      961 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1021 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     1081 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1141 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1201 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1261 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1321 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1381 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     1441 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     1501 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     1561 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     1621 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     1681 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     1741 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     1801 atcgtttcag aatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg
     1861 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     1921 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     1981 gcagagatcc actttggcgc cggctcgagg gggcccgggt gcaaagatgg ataaagtttt
     2041 aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga gtgggaattg
     2101 gctccgtagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc
     2161 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag
     2221 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
     2281 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
     2341 gggtttgccg ccagaacaca ggaccggttc tagagcgctg ccaccatgga caagaagtac
     2401 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     2461 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     2521 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     2581 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     2641 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     2701 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     2761 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     2821 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     2881 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     2941 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     3001 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     3061 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     3121 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     3181 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     3241 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     3301 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     3361 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     3421 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     3481 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     3541 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     3601 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     3661 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     3721 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     3781 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     3841 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     3901 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     3961 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     4021 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     4081 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     4141 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     4201 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     4261 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     4321 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     4381 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     4441 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     4501 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     4561 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     4621 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     4681 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     4741 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     4801 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     4861 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     4921 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     4981 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     5041 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     5101 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     5161 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     5221 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     5281 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     5341 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     5401 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     5461 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     5521 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     5581 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     5641 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     5701 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     5761 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     5821 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     5881 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     5941 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     6001 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     6061 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     6121 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     6181 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     6241 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     6301 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     6361 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     6421 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
     6481 ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa gaagaaagat
     6541 tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct gaaacaagcc
     6601 ggagatgtcg aagagaatcc tggaccgatg accgagtaca agcccacggt gcgcctcgcc
     6661 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     6721 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     6781 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     6841 gccgtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     6901 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     6961 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg
     7021 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     7081 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     7141 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     7201 tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac
     7261 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     7321 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     7381 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     7441 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     7501 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     7561 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     7621 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     7681 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     7741 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     7801 agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg
     7861 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     7921 cccaacgaag acaagatttc accattatcg tttcagaccc acctcccaac cccgagggga
     7981 cccagagagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     8041 agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     8101 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     8161 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     8221 aaaggacgaa acaccgtggc cttcacaacc aaagcagttt tagagctaga aatagcaagt
     8281 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttaa
     8341 gcttggcgta actagatctt gagacactgc tttttgcttg tactgggtct ctctggttag
     8401 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     8461 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     8521 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt
     8581 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     8641 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     8701 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     8761 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
     8821 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     8881 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     8941 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     9001 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     9061 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     9121 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     9181 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     9241 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     9301 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     9361 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     9421 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     9481 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     9541 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     9601 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     9661 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     9721 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     9781 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     9841 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     9901 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     9961 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
    10021 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
    10081 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
    10141 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
    10201 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
    10261 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
    10321 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
    10381 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
    10441 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
    10501 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
    10561 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
    10621 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
    10681 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
    10741 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
    10801 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
    10861 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
    10921 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
    10981 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
    11041 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
    11101 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
    11161 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
    11221 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
    11281 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
    11341 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
    11401 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
    11461 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
    11521 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
    11581 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
    11641 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt c
//
