LOCUS       sequence-362623-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets ERBB2 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_ERBB2
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     31..51
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        33..51
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        79..305
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             306..486
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    533..658
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1151..1384
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1569..1613
                     /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    1794..1911
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2100..2311
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2330..2339
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2336..6439
                     /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             2336..6439
                     /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             6440..6487
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6488..6511
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6521..6577
                     /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             6578..7177
                     /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(6578..6597)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7074..7094
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7193..7781
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7246..7266)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7664..7675)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7937..8185
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7937..7957
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8108..8127
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8207..8282
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8335..8515
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8593..8714
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8630..8649)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8684..8703
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8754..8889
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8909..8928)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8910..8928)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8938..8955)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8938..8954)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8947..8969)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9096..9551
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9183..9202)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9393..9414
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9577..9681
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9682..9750,9751..10542)
                     /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(9900..9919)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10713..11301
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11202..11221
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11455..11472
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11589..11610
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11625..11630,11631..11648,11649..11655)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11663..11679
                     /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     11668..11690
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(11687..11691,1..12)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(11687..11691,1..12)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
       61 ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa
      121 cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg
      181 aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg
      241 gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca
      301 taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
      361 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
      421 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
      481 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg
      541 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag
      601 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag
      661 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa
      721 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt
      781 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc
      841 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta
      901 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga
      961 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag
     1021 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
     1081 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
     1141 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
     1201 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
     1261 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
     1321 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
     1381 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg
     1441 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg
     1501 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc
     1561 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt
     1621 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct
     1681 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
     1741 attcaccatt atcgtttcag aatcttgaga caaatggcag tattcatcca caattttaaa
     1801 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     1861 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     1921 tacagggaca gcagagatcc actttggcgc cggctcgagg gggcccgggt gcaaagatgg
     1981 ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga
     2041 gtgggaattg gctccgtagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg
     2101 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgatc
     2161 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     2221 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     2281 ttttcgcaac gggtttgccg ccagaacaca ggaccggttc tagagcgctg ccaccatgga
     2341 caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac
     2401 cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag
     2461 catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac
     2521 ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct
     2581 gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga
     2641 agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat
     2701 cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact
     2761 ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat
     2821 caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga
     2881 caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa
     2941 cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct
     3001 ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat
     3061 tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc
     3121 caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat
     3181 cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct
     3241 gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat
     3301 caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca
     3361 gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta
     3421 cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa
     3481 gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca
     3541 gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat
     3601 tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa
     3661 gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt
     3721 cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt
     3781 ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct
     3841 gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa
     3901 cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg
     3961 cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa
     4021 gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg
     4081 cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa
     4141 ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac
     4201 cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct
     4261 gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct
     4321 gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt
     4381 cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct
     4441 gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga
     4501 gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa
     4561 ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga
     4621 aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa
     4681 gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga
     4741 aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat
     4801 gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt
     4861 gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa
     4921 gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta
     4981 ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa
     5041 ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt
     5101 ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa
     5161 gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct
     5221 ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca
     5281 ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc
     5341 taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat
     5401 cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat
     5461 catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct
     5521 gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac
     5581 cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac
     5641 aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag
     5701 aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc
     5761 tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga
     5821 gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct
     5881 ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc
     5941 cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa
     6001 gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta
     6061 tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca
     6121 caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct
     6181 ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat
     6241 cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc
     6301 cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt
     6361 gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct
     6421 gtctcagctg ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa
     6481 gaagaaagat tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct
     6541 gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg accgagtaca agcccacggt
     6601 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     6661 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     6721 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     6781 cgacggcgcc gccgtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     6841 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     6901 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     6961 cggagtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     7021 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     7081 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     7141 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc
     7201 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     7261 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     7321 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     7381 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     7441 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     7501 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     7561 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     7621 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     7681 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     7741 ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg
     7801 acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg
     7861 ctaattcact cccaacgaag acaagatttc accattatcg tttcagaccc acctcccaac
     7921 cccgagggga cccagagagg gcctatttcc catgattcct tcatatttgc atatacgata
     7981 caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca
     8041 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     8101 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     8161 tatcttgtgg aaaggacgaa acaccgcaga acctgcaagt aatccggttt tagagctaga
     8221 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     8281 gcttttttaa gcttggcgta actagatctt gagacactgc tttttgcttg tactgggtct
     8341 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     8401 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     8461 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg
     8521 tatagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca
     8581 gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     8641 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     8701 atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc
     8761 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     8821 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     8881 ttggaggcct agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     8941 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     9001 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     9061 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     9121 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     9181 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     9241 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     9301 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     9361 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     9421 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     9481 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     9541 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     9601 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     9661 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     9721 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     9781 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     9841 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     9901 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     9961 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
    10021 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
    10081 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
    10141 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
    10201 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
    10261 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
    10321 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
    10381 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
    10441 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
    10501 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
    10561 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
    10621 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
    10681 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
    10741 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
    10801 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
    10861 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
    10921 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
    10981 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
    11041 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
    11101 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
    11161 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
    11221 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
    11281 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
    11341 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
    11401 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
    11461 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
    11521 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
    11581 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
    11641 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg a
//
