LOCUS       sequence-362621-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets EGFR 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_EGFR
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..48
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        30..48
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        76..302
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             303..483
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    530..655
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1148..1381
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1566..1610
                     /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    1791..1908
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2097..2308
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2327..2336
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2333..6436
                     /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             2333..6436
                     /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             6437..6484
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6485..6508
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6518..6574
                     /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             6575..7174
                     /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(6575..6594)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7071..7091
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7190..7778
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7243..7263)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7661..7672)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7934..8182
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7934..7954
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8105..8124
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8204..8279
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8332..8512
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8590..8711
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8627..8646)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8681..8700
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8751..8886
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8906..8925)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8907..8925)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8935..8952)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8935..8951)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8944..8966)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9093..9548
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9180..9199)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9390..9411
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9574..9678
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9679..9747,9748..10539)
                     /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(9897..9916)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10710..11298
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11199..11218
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11452..11469
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11586..11607
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11622..11627,11628..11645,11646..11652)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11660..11676
                     /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     11665..11687
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(11684..11691,1..9)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(11684..11691,1..9)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
       61 gagctgcaag cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga
      121 tgagttagca acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag
      181 taaggtggta cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac
      241 gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa
      301 acgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
      361 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
      421 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
      481 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg
      541 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac
      601 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat
      661 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa
      721 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
      781 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc
      841 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
      901 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga
      961 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag
     1021 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac
     1081 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag
     1141 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag
     1201 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga
     1261 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca
     1321 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc
     1381 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta
     1441 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca
     1501 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc
     1561 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt
     1621 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg
     1681 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt
     1741 caccattatc gtttcagaat cttgagacaa atggcagtat tcatccacaa ttttaaaaga
     1801 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     1861 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     1921 agggacagca gagatccact ttggcgccgg ctcgaggggg cccgggtgca aagatggata
     1981 aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt gaaaggagtg
     2041 ggaattggct ccgtaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
     2101 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg
     2161 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     2221 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     2281 tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa
     2341 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
     2401 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     2461 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     2521 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     2581 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     2641 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     2701 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     2761 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     2821 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     2881 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     2941 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     3001 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     3061 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     3121 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     3181 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     3241 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     3301 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     3361 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     3421 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     3481 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     3541 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     3601 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     3661 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     3721 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     3781 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     3841 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     3901 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     3961 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     4021 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     4081 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     4141 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     4201 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     4261 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     4321 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     4381 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     4441 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     4501 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     4561 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     4621 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     4681 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     4741 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     4801 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     4861 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     4921 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     4981 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     5041 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     5101 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     5161 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     5221 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     5281 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     5341 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     5401 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     5461 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     5521 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     5581 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     5641 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     5701 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     5761 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     5821 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     5881 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     5941 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     6001 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     6061 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     6121 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     6181 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     6241 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     6301 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     6361 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     6421 tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa
     6481 gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa
     6541 acaagccgga gatgtcgaag agaatcctgg accgatgacc gagtacaagc ccacggtgcg
     6601 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     6661 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     6721 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     6781 cggcgccgcc gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     6841 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     6901 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     6961 agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     7021 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     7081 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     7141 cacctggtgc atgacccgca agcccggtgc ctgaacgcgt taagtcgaca atcaacctct
     7201 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     7261 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     7321 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     7381 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     7441 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     7501 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     7561 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     7621 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     7681 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     7741 tcagacgagt cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact
     7801 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     7861 attcactccc aacgaagaca agatttcacc attatcgttt cagacccacc tcccaacccc
     7921 gaggggaccc agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     7981 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     8041 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     8101 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     8161 cttgtggaaa ggacgaaaca ccgctcttct tagaccatcc agggttttag agctagaaat
     8221 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     8281 tttttaagct tggcgtaact agatcttgag acactgcttt ttgcttgtac tgggtctctc
     8341 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     8401 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     8461 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat
     8521 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     8581 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     8641 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     8701 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
     8761 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     8821 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     8881 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     8941 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     9001 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     9061 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     9121 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     9181 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     9241 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     9301 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     9361 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     9421 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     9481 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     9541 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     9601 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     9661 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     9721 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     9781 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     9841 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     9901 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     9961 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
    10021 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
    10081 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
    10141 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
    10201 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
    10261 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
    10321 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
    10381 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
    10441 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
    10501 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
    10561 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
    10621 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
    10681 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
    10741 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
    10801 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
    10861 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
    10921 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
    10981 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
    11041 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
    11101 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
    11161 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
    11221 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
    11281 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
    11341 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
    11401 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
    11461 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
    11521 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
    11581 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
    11641 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa c
//
