LOCUS       sequence-362613-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets CSF3R 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_CSF3R
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..29
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13..29
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     48..68
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        50..68
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        96..322
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             323..503
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    550..675
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1168..1401
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1586..1630
                     /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    1811..1928
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2117..2328
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2347..2356
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2353..6456
                     /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             2353..6456
                     /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             6457..6504
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6505..6528
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6538..6594
                     /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             6595..7194
                     /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(6595..6614)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7091..7111
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7210..7798
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7263..7283)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7681..7692)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7954..8202
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7954..7974
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8125..8144
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8224..8299
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8352..8532
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8610..8731
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8647..8666)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8701..8720
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8771..8906
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8926..8945)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8927..8945)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8955..8972)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8955..8971)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8964..8986)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9113..9568
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9200..9219)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9410..9431
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9594..9698
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9699..9767,9768..10559)
                     /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(9917..9936)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10730..11318
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11219..11238
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11472..11489
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11606..11627
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11642..11647,11648..11665,11666..11672)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(11680..11691,1..5)
                     /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     join(11685..11691,1..16)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
       61 actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta
      121 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg
      181 tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac
      241 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa
      301 gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct
      361 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
      421 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
      481 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa
      541 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag
      601 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat
      661 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg
      721 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag
      781 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata
      841 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat
      901 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct
      961 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct
     1021 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     1081 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     1141 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     1201 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     1261 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     1321 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     1381 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     1441 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     1501 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     1561 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     1621 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     1681 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     1741 tagagttagg cagggatatt caccattatc gtttcagaat cttgagacaa atggcagtat
     1801 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
     1861 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
     1921 aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg ctcgaggggg
     1981 cccgggtgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct
     2041 tctaggtctt gaaaggagtg ggaattggct ccgtaggtct tgaaaggagt gggaattggc
     2101 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     2161 ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     2221 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     2281 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga ccggttctag
     2341 agcgctgcca ccatggacaa gaagtacagc atcggcctgg acatcggcac caactctgtg
     2401 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
     2461 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
     2521 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
     2581 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
     2641 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
     2701 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
     2761 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
     2821 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
     2881 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
     2941 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
     3001 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
     3061 ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
     3121 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
     3181 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
     3241 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
     3301 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
     3361 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
     3421 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
     3481 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
     3541 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
     3601 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
     3661 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
     3721 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
     3781 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
     3841 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
     3901 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
     3961 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
     4021 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
     4081 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
     4141 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
     4201 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
     4261 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
     4321 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
     4381 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
     4441 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
     4501 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
     4561 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
     4621 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
     4681 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
     4741 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
     4801 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
     4861 gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg
     4921 ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg
     4981 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
     5041 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
     5101 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
     5161 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
     5221 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
     5281 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
     5341 ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac
     5401 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
     5461 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
     5521 atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
     5581 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
     5641 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
     5701 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
     5761 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
     5821 ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag
     5881 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
     5941 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
     6001 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
     6061 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
     6121 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
     6181 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
     6241 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
     6301 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
     6361 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
     6421 gagacacgga tcgacctgtc tcagctggga ggcgacaagc gacctgccgc cacaaagaag
     6481 gctggacagg ctaagaagaa gaaagattac aaagacgatg acgataaggg atccggcgca
     6541 acaaacttct ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accgatgacc
     6601 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     6661 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     6721 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     6781 aaggtgtggg tcgcggacga cggcgccgcc gtggcggtct ggaccacgcc ggagagcgtc
     6841 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     6901 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     6961 tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     7021 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     7081 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     7141 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaacgcgt
     7201 taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     7261 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     7321 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     7381 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     7441 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     7501 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     7561 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     7621 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     7681 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     7741 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgt
     7801 cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     7861 aggggggact ggaagggcta attcactccc aacgaagaca agatttcacc attatcgttt
     7921 cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat gattccttca
     7981 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
     8041 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     8101 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     8161 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggtgcccc tggaggaaga
     8221 caggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     8281 agtggcaccg agtcggtgct tttttaagct tggcgtaact agatcttgag acactgcttt
     8341 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     8401 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     8461 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     8521 aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg
     8581 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     8641 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     8701 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     8761 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     8821 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     8881 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     8941 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     9001 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     9061 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     9121 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     9181 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     9241 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     9301 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     9361 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     9421 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     9481 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     9541 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     9601 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     9661 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     9721 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     9781 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     9841 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     9901 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     9961 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
    10021 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
    10081 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
    10141 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
    10201 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
    10261 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
    10321 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
    10381 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
    10441 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
    10501 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
    10561 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
    10621 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
    10681 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
    10741 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
    10801 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
    10861 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
    10921 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
    10981 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
    11041 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
    11101 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
    11161 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
    11221 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
    11281 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
    11341 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
    11401 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
    11461 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
    11521 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
    11581 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
    11641 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga t
//
