LOCUS       sequence-362909-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets the AAVS1 
            locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  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 13013)
  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_AAVS1D
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        93..140
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             159..533
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    663..784
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(700..719)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     754..773
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(833..849)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(833..849)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(846..868)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    857..873
                     /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)."
     promoter        complement(join(881..887,888..905,906..911))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    926..947
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1064..1081)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1235..1823)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1315..1334)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1994..2785,2786..2854))
                     /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     2617..2636
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2855..2959)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3034..3053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3225..3604
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3606..3804
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3759..3779
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3822..4002
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4049..4174
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4667..4900
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5085..5129
                     /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    5427..5544
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5595..5843
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5595..5615
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5766..5785
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5865..5940
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6002..6213
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6232..6241
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6238..10341
                     /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             6238..10341
                     /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             10342..10389
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10390..10413
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             10423..10479
                     /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             10480..11076
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     10973..10993
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    11092..11680
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11145..11165)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11563..11574)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             11752..11985
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12011..12028)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12017..12241
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12287..12715
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12374..12393)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12584..12605
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12724..12744)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(12729..13013,1..45)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(12909..13013,1..31)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12971..12990
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
       61 tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata
      121 tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg
      181 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg
      241 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc
      301 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg
      361 tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg
      421 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc
      481 tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc
      541 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
      601 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
      661 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
      721 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
      781 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
      841 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
      901 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
      961 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1021 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     1081 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     1141 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     1201 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     1261 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     1321 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     1381 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     1441 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1501 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     1561 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     1621 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     1681 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     1741 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     1801 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     1861 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     1921 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     1981 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     2041 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     2101 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     2161 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     2221 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     2281 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     2341 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     2401 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     2461 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     2521 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     2581 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     2641 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     2701 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     2761 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     2821 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     2881 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     2941 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     3001 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3061 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     3121 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     3181 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     3241 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     3301 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     3361 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     3421 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     3481 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     3541 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     3601 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     3661 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     3721 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     3781 acggtgggag gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc
     3841 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     3901 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     3961 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     4021 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     4081 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     4141 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     4201 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     4261 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     4321 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     4381 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     4441 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     4501 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     4561 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     4621 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     4681 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     4741 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     4801 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     4861 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     4921 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     4981 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     5041 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     5101 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     5161 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     5221 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     5281 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     5341 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     5401 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     5461 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     5521 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     5581 ttaattaagg taccgagggc ctatttccca tgattccttc atatttgcat atacgataca
     5641 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
     5701 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     5761 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     5821 tcttgtggaa aggacgaaac accgggccat tcccggcctc cctggtttta gagctagaaa
     5881 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     5941 ttttttgaat tcgctagcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
     6001 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     6061 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     6121 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     6181 ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagagcgc tgccaccatg
     6241 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
     6301 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     6361 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
     6421 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
     6481 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     6541 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     6601 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     6661 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     6721 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     6781 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     6841 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     6901 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
     6961 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     7021 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     7081 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     7141 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     7201 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     7261 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     7321 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     7381 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     7441 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     7501 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     7561 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     7621 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     7681 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     7741 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     7801 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     7861 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     7921 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     7981 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     8041 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     8101 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     8161 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     8221 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     8281 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     8341 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     8401 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     8461 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     8521 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     8581 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     8641 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     8701 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     8761 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     8821 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     8881 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     8941 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     9001 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     9061 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     9121 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     9181 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     9241 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     9301 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     9361 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     9421 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     9481 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     9541 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     9601 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     9661 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     9721 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     9781 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     9841 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     9901 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     9961 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
    10021 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
    10081 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
    10141 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
    10201 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
    10261 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
    10321 ctgtctcagc tgggaggcga caagcgacct gccgccacaa agaaggctgg acaggctaag
    10381 aagaagaaag attacaaaga cgatgacgat aagggatccg gcgcaacaaa cttctctctg
    10441 ctgaaacaag ccggagatgt cgaagagaat cctggaccga ccgagtacaa gcccacggtg
    10501 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
    10561 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
    10621 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
    10681 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
    10741 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
    10801 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
    10861 ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
    10921 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
    10981 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
    11041 cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct
    11101 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
    11161 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
    11221 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
    11281 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
    11341 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
    11401 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
    11461 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
    11521 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
    11581 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
    11641 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
    11701 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
    11761 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
    11821 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
    11881 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
    11941 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc
    12001 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
    12061 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
    12121 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
    12181 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
    12241 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
    12301 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
    12361 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
    12421 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
    12481 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
    12541 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
    12601 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
    12661 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
    12721 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
    12781 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
    12841 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
    12901 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
    12961 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttc
//
