LOCUS       sequence-362765-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets TOP2B 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_TOP2B
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..26
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     23..39
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     23..39
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     58..78
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        60..78
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        106..332
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             333..513
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    560..685
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1178..1411
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1596..1640
                     /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    1821..1938
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2127..2338
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2357..2366
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2363..6466
                     /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             2363..6466
                     /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             6467..6514
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6515..6538
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6548..6604
                     /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             6605..7204
                     /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(6605..6624)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7101..7121
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7220..7808
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7273..7293)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7691..7702)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7964..8212
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7964..7984
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8135..8154
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8234..8309
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8362..8542
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8620..8741
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8657..8676)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8711..8730
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8781..8916
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8936..8955)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8937..8955)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8965..8982)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8965..8981)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8974..8996)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9123..9578
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9210..9229)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9420..9441
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9604..9708
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9709..9777,9778..10569)
                     /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(9927..9946)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10740..11328
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11229..11248
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11482..11499
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11616..11637
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11652..11657,11658..11675,11676..11682)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(11690..11691,1..15)
                     /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)."
ORIGIN
        1 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
       61 attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa
      121 tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga
      181 aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa
      241 ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata
      301 ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag
      361 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      421 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      481 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      541 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      601 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      661 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      721 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
      781 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
      841 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
      901 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
      961 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1021 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     1081 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     1141 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     1201 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     1261 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     1321 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     1381 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     1441 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     1501 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     1561 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     1621 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     1681 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     1741 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagaat cttgagacaa
     1801 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     1861 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     1921 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg
     1981 ctcgaggggg cccgggtgca aagatggata aagttttaaa cagagaggaa tctttgcagc
     2041 taatggacct tctaggtctt gaaaggagtg ggaattggct ccgtaggtct tgaaaggagt
     2101 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     2161 gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg
     2221 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     2281 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga
     2341 ccggttctag agcgctgcca ccatggacaa gaagtacagc atcggcctgg acatcggcac
     2401 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     2461 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
     2521 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     2581 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
     2641 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     2701 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     2761 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     2821 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     2881 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     2941 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     3001 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     3061 gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     3121 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     3181 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     3241 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     3301 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     3361 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     3421 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     3481 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     3541 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     3601 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     3661 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     3721 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     3781 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     3841 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     3901 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     3961 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt
     4021 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
     4081 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
     4141 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
     4201 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
     4261 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
     4321 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
     4381 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
     4441 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
     4501 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
     4561 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
     4621 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
     4681 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
     4741 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
     4801 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
     4861 gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga
     4921 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga
     4981 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
     5041 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
     5101 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
     5161 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
     5221 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
     5281 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
     5341 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
     5401 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
     5461 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
     5521 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
     5581 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
     5641 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
     5701 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
     5761 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
     5821 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
     5881 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
     5941 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
     6001 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
     6061 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
     6121 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
     6181 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
     6241 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
     6301 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
     6361 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
     6421 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaagc gacctgccgc
     6481 cacaaagaag gctggacagg ctaagaagaa gaaagattac aaagacgatg acgataaggg
     6541 atccggcgca acaaacttct ctctgctgaa acaagccgga gatgtcgaag agaatcctgg
     6601 accgatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     6661 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     6721 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     6781 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcc gtggcggtct ggaccacgcc
     6841 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     6901 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     6961 ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct
     7021 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     7081 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     7141 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     7201 ctgaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     7261 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     7321 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     7381 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     7441 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     7501 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     7561 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     7621 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     7681 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     7741 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     7801 ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     7861 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatttcacc
     7921 attatcgttt cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat
     7981 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt
     8041 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     8101 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     8161 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgtgtagga
     8221 atgaattgca ggggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     8281 aacttgaaaa agtggcaccg agtcggtgct tttttaagct tggcgtaact agatcttgag
     8341 acactgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     8401 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     8461 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     8521 tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat
     8581 ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat
     8641 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     8701 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta
     8761 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     8821 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     8881 tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta
     8941 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     9001 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     9061 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     9121 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     9181 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     9241 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     9301 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     9361 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     9421 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     9481 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     9541 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     9601 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     9661 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     9721 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     9781 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     9841 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     9901 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     9961 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
    10021 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
    10081 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
    10141 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
    10201 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
    10261 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
    10321 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
    10381 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
    10441 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
    10501 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
    10561 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
    10621 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
    10681 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
    10741 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
    10801 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
    10861 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
    10921 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
    10981 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
    11041 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
    11101 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
    11161 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
    11221 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
    11281 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
    11341 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
    11401 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
    11461 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
    11521 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
    11581 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
    11641 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat t
//
