LOCUS       sequence-362637-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets IDH1 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_IDH1
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..50
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        32..50
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        78..304
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             305..485
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    532..657
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1150..1383
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1568..1612
                     /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    1793..1910
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2099..2310
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2329..2338
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2335..6438
                     /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             2335..6438
                     /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             6439..6486
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6487..6510
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6520..6576
                     /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             6577..7176
                     /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(6577..6596)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7073..7093
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7192..7780
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7245..7265)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7663..7674)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7936..8184
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7936..7956
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8107..8126
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8206..8281
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8334..8514
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8592..8713
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8629..8648)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8683..8702
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8753..8888
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8908..8927)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8909..8927)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8937..8954)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8937..8953)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8946..8968)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9095..9550
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9182..9201)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9392..9413
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9576..9680
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9681..9749,9750..10541)
                     /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(9899..9918)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10712..11300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11201..11220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11454..11471
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11588..11609
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11624..11629,11630..11647,11648..11654)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11662..11678
                     /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     11667..11689
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(11686..11691,1..11)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(11686..11691,1..11)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
       61 tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac
      121 gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga
      181 agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg
      241 acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat
      301 aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
      361 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
      421 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
      481 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
      541 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
      601 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
      661 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
      721 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
      781 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
      841 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
      901 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
      961 gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg
     1021 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
     1081 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
     1141 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
     1201 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
     1261 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
     1321 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
     1381 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
     1441 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
     1501 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
     1561 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
     1621 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
     1681 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
     1741 ttcaccatta tcgtttcaga atcttgagac aaatggcagt attcatccac aattttaaaa
     1801 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     1861 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     1921 acagggacag cagagatcca ctttggcgcc ggctcgaggg ggcccgggtg caaagatgga
     1981 taaagtttta aacagagagg aatctttgca gctaatggac cttctaggtc ttgaaaggag
     2041 tgggaattgg ctccgtaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg
     2101 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgatcc
     2161 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     2221 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     2281 tttcgcaacg ggtttgccgc cagaacacag gaccggttct agagcgctgc caccatggac
     2341 aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc
     2401 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
     2461 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
     2521 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
     2581 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
     2641 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
     2701 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
     2761 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
     2821 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
     2881 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
     2941 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
     3001 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt
     3061 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
     3121 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
     3181 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
     3241 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
     3301 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
     3361 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
     3421 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
     3481 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
     3541 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
     3601 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
     3661 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
     3721 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
     3781 gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg
     3841 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
     3901 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
     3961 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
     4021 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
     4081 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
     4141 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
     4201 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
     4261 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
     4321 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
     4381 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
     4441 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
     4501 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
     4561 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
     4621 atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag
     4681 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
     4741 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
     4801 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg
     4861 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
     4921 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
     4981 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
     5041 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
     5101 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
     5161 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
     5221 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
     5281 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
     5341 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
     5401 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
     5461 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
     5521 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
     5581 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
     5641 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
     5701 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
     5761 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
     5821 ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
     5881 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
     5941 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
     6001 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
     6061 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
     6121 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
     6181 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
     6241 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
     6301 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
     6361 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
     6421 tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga aggctggaca ggctaagaag
     6481 aagaaagatt acaaagacga tgacgataag ggatccggcg caacaaactt ctctctgctg
     6541 aaacaagccg gagatgtcga agagaatcct ggaccgatga ccgagtacaa gcccacggtg
     6601 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     6661 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     6721 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     6781 gacggcgccg ccgtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     6841 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     6901 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     6961 ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     7021 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     7081 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     7141 cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct
     7201 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     7261 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     7321 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     7381 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     7441 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     7501 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     7561 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     7621 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     7681 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     7741 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
     7801 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     7861 taattcactc ccaacgaaga caagatttca ccattatcgt ttcagaccca cctcccaacc
     7921 ccgaggggac ccagagaggg cctatttccc atgattcctt catatttgca tatacgatac
     7981 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
     8041 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     8101 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     8161 atcttgtgga aaggacgaaa caccgaccca tccactcaca agccggtttt agagctagaa
     8221 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     8281 cttttttaag cttggcgtaa ctagatcttg agacactgct ttttgcttgt actgggtctc
     8341 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     8401 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     8461 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt
     8521 atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     8581 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     8641 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     8701 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc
     8761 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     8821 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     8881 tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     8941 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     9001 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     9061 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     9121 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     9181 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     9241 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     9301 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     9361 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     9421 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     9481 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     9541 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     9601 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     9661 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     9721 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     9781 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     9841 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     9901 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     9961 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
    10021 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
    10081 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
    10141 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
    10201 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
    10261 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
    10321 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
    10381 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
    10441 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
    10501 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
    10561 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
    10621 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
    10681 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
    10741 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
    10801 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
    10861 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
    10921 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
    10981 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
    11041 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
    11101 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
    11161 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
    11221 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
    11281 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
    11341 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
    11401 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
    11461 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
    11521 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
    11581 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
    11641 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga a
//
