LOCUS       sequence-362907-       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_AAVS1B
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(605..1396,1397..1465))
                     /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     1228..1247
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1466..1570)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1645..1664)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1836..2215
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2217..2415
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2370..2390
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2433..2613
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2660..2785
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3278..3511
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3696..3740
                     /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    4038..4155
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4206..4454
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4206..4226
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4377..4396
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4476..4551
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4613..4824
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4843..4852
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4849..8952
                     /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             4849..8952
                     /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             8953..9000
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9001..9024
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             9034..9090
                     /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             9091..9687
                     /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     9584..9604
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9703..10291
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9756..9776)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10174..10185)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10363..10596
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10622..10639)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10628..10852
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10898..11326
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10985..11004)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11195..11216
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11335..11355)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11340..11669
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11520..11655
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11582..11601
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11717..11764
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11783..12157
                     /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    12287..12408
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12324..12343)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12378..12397
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12457..12473)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12457..12473)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12470..12492)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12481..12497
                     /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(12505..12511,12512..12529,12530..12535))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12550..12571
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12688..12705)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(12859..13013,1..434))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12939..12958)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
       61 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      121 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      181 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      241 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      301 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      361 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      421 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      481 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
      541 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
      601 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
      661 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
      721 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
      781 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
      841 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
      901 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
      961 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1021 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1081 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1141 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1201 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1261 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1321 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     1381 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     1441 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     1501 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     1561 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     1621 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     1681 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     1741 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     1801 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     1861 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     1921 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     1981 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     2041 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     2101 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     2161 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     2221 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     2281 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     2341 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     2401 ggtctatata agcagcgcgt tttgcctgta ctgggtctct ctggttagac cagatctgag
     2461 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     2521 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     2581 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
     2641 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
     2701 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
     2761 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
     2821 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
     2881 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
     2941 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     3001 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     3061 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     3121 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     3181 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     3241 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     3301 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     3361 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     3421 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     3481 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     3541 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     3601 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     3661 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     3721 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     3781 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     3841 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     3901 ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag
     3961 acagatccat tcgattagtg aacggatcgg cactgcgtgc gccaattctg cagacaaatg
     4021 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     4081 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     4141 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gttaattaag
     4201 gtaccgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     4261 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     4321 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     4381 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     4441 aaggacgaaa caccgggtcc aaacttaggg atgtggtttt agagctagaa atagcaagtt
     4501 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgaa
     4561 ttcgctagct aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag
     4621 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc
     4681 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     4741 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     4801 aacgggtttg ccgccagaac acaggaccgg ttctagagcg ctgccaccat ggacaagaag
     4861 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     4921 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     4981 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     5041 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     5101 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     5161 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     5221 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     5281 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     5341 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     5401 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     5461 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     5521 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     5581 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     5641 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     5701 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     5761 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     5821 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     5881 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     5941 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     6001 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     6061 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     6121 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     6181 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     6241 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     6301 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     6361 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     6421 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     6481 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     6541 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     6601 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     6661 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     6721 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     6781 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     6841 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     6901 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     6961 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     7021 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     7081 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     7141 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     7201 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     7261 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     7321 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     7381 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     7441 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     7501 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     7561 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     7621 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     7681 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     7741 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     7801 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     7861 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     7921 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     7981 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     8041 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     8101 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     8161 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     8221 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     8281 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     8341 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     8401 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     8461 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     8521 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     8581 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     8641 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     8701 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     8761 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     8821 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     8881 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     8941 ctgggaggcg acaagcgacc tgccgccaca aagaaggctg gacaggctaa gaagaagaaa
     9001 gattacaaag acgatgacga taagggatcc ggcgcaacaa acttctctct gctgaaacaa
     9061 gccggagatg tcgaagagaa tcctggaccg accgagtaca agcccacggt gcgcctcgcc
     9121 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     9181 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     9241 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     9301 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     9361 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     9421 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg
     9481 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     9541 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     9601 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     9661 tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac
     9721 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     9781 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     9841 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     9901 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     9961 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
    10021 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
    10081 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
    10141 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
    10201 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
    10261 agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg
    10321 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
    10381 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
    10441 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
    10501 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
    10561 tcagaccctt ttagtcagtg tggaaaatct ctagcagggc ccgtttaaac ccgctgatca
    10621 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
    10681 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
    10741 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
    10801 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
    10861 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
    10921 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
    10981 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
    11041 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
    11101 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
    11161 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
    11221 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
    11281 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
    11341 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
    11401 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
    11461 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
    11521 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
    11581 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
    11641 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
    11701 gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata
    11761 cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc
    11821 gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt
    11881 cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt
    11941 ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga
    12001 gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc
    12061 catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg
    12121 caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc
    12181 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
    12241 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
    12301 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
    12361 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
    12421 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
    12481 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
    12541 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
    12601 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
    12661 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
    12721 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
    12781 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
    12841 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
    12901 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
    12961 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acc
//
