LOCUS       sequence-362768-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets MYC 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_MYC
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..21
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5..21
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     40..60
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        42..60
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        88..314
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             315..495
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    542..667
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1160..1393
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1578..1622
                     /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    1803..1920
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2109..2320
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2339..2348
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2345..6448
                     /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             2345..6448
                     /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             6449..6496
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6497..6520
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6530..6586
                     /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             6587..7186
                     /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(6587..6606)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7083..7103
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7202..7790
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7255..7275)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7673..7684)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7946..8194
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7946..7966
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8117..8136
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8216..8291
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8344..8524
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8602..8723
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8639..8658)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8693..8712
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8763..8898
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8918..8937)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8919..8937)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8947..8964)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8947..8963)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8956..8978)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9105..9560
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9192..9211)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9402..9423
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9586..9690
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9691..9759,9760..10551)
                     /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(9909..9928)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10722..11310
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11211..11230
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11464..11481
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11598..11619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11634..11639,11640..11657,11658..11664)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11672..11688
                     /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(11677..11691,1..8)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
       61 gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca
      121 acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc
      181 gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga
      241 catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag
      301 ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct
      361 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
      421 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
      481 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
      541 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
      601 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
      661 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
      721 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
      781 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
      841 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
      901 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
      961 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca
     1021 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     1081 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     1141 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     1201 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     1261 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     1321 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     1381 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     1441 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     1501 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     1561 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     1621 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     1681 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     1741 ggcagggata ttcaccatta tcgtttcaga atcttgagac aaatggcagt attcatccac
     1801 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     1861 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     1921 cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgaggg ggcccgggtg
     1981 caaagatgga taaagtttta aacagagagg aatctttgca gctaatggac cttctaggtc
     2041 ttgaaaggag tgggaattgg ctccgtaggt cttgaaagga gtgggaattg gctccggtgc
     2101 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     2161 caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     2221 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     2281 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gaccggttct agagcgctgc
     2341 caccatggac aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc
     2401 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     2461 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc
     2521 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
     2581 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
     2641 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
     2701 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     2761 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     2821 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     2881 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     2941 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     3001 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     3061 aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     3121 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     3181 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     3241 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     3301 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     3361 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     3421 cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     3481 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     3541 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     3601 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     3661 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     3721 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     3781 ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga
     3841 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     3901 cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     3961 cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt
     4021 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     4081 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     4141 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     4201 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     4261 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     4321 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     4381 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     4441 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     4501 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     4561 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     4621 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     4681 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     4741 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     4801 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     4861 ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag
     4921 aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     4981 gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa
     5041 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     5101 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     5161 gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa
     5221 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     5281 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     5341 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     5401 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     5461 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     5521 gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga
     5581 ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga
     5641 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct
     5701 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
     5761 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     5821 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     5881 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     5941 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
     6001 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
     6061 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
     6121 ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag
     6181 agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga
     6241 taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
     6301 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
     6361 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
     6421 gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga aggctggaca
     6481 ggctaagaag aagaaagatt acaaagacga tgacgataag ggatccggcg caacaaactt
     6541 ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgatga ccgagtacaa
     6601 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     6661 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     6721 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     6781 ggtcgcggac gacggcgccg ccgtggcggt ctggaccacg ccggagagcg tcgaagcggg
     6841 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     6901 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     6961 ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     7021 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     7081 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     7141 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga
     7201 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     7261 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     7321 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     7381 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     7441 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     7501 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     7561 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     7621 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     7681 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     7741 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta
     7801 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     7861 ctggaagggc taattcactc ccaacgaaga caagatttca ccattatcgt ttcagaccca
     7921 cctcccaacc ccgaggggac ccagagaggg cctatttccc atgattcctt catatttgca
     7981 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
     8041 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     8101 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     8161 ggctttatat atcttgtgga aaggacgaaa caccgcttcg gggagacaac gacgggtttt
     8221 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     8281 cgagtcggtg cttttttaag cttggcgtaa ctagatcttg agacactgct ttttgcttgt
     8341 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     8401 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     8461 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     8521 agcagtacgt atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     8581 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     8641 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     8701 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     8761 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     8821 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     8881 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc
     8941 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     9001 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     9061 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     9121 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     9181 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     9241 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     9301 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     9361 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     9421 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     9481 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     9541 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     9601 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     9661 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     9721 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     9781 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     9841 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     9901 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     9961 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
    10021 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
    10081 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
    10141 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
    10201 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
    10261 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
    10321 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
    10381 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
    10441 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
    10501 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
    10561 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
    10621 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
    10681 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
    10741 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
    10801 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
    10861 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
    10921 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
    10981 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
    11041 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
    11101 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
    11161 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
    11221 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
    11281 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
    11341 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
    11401 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
    11461 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
    11521 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
    11581 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
    11641 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt t
//
