LOCUS       sequence-362797-       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_AAVS1A
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      23..158
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     85..104
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        220..267
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             286..660
                     /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    790..911
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(827..846)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     881..900
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(960..976)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(960..976)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(973..995)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    984..1000
                     /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(1008..1014,1015..1032,1033..1038))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1053..1074
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1191..1208)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1362..1950)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1442..1461)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2121..2912,2913..2981))
                     /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     2744..2763
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2982..3086)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3161..3180)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3352..3731
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3733..3931
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3886..3906
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3949..4129
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4176..4301
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4794..5027
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5212..5256
                     /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    5554..5671
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5722..5970
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5722..5742
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5893..5912
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5992..6067
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6129..6340
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6359..6368
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6365..10468
                     /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             6365..10468
                     /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             10469..10516
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10517..10540
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             10550..10606
                     /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             10607..11203
                     /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     11100..11120
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    11219..11807
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11272..11292)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11690..11701)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             11879..12112
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12138..12155)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12144..12368
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12414..12842
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12501..12520)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12711..12732
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12851..12871)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(12856..13013,1..172)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
       61 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
      121 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
      181 gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca
      241 tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc
      301 agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac
      361 cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac
      421 gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg
      481 gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac
      541 ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag
      601 ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga
      661 cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
      721 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
      781 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
      841 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
      901 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
      961 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     1021 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     1081 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     1141 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     1201 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     1261 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     1321 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1381 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1441 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1501 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1561 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1621 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1681 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1741 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     1801 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1861 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1921 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1981 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     2041 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     2101 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     2161 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     2221 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     2281 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     2341 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     2401 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     2461 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     2521 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     2581 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     2641 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     2701 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     2761 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     2821 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     2881 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     2941 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     3001 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     3061 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     3121 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     3181 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     3241 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     3301 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
     3361 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     3421 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     3481 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     3541 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     3601 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     3661 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     3721 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     3781 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     3841 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     3901 ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg
     3961 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     4021 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     4081 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
     4141 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
     4201 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
     4261 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
     4321 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
     4381 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
     4441 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     4501 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     4561 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     4621 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     4681 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     4741 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     4801 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     4861 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     4921 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     4981 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     5041 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     5101 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     5161 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     5221 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     5281 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     5341 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     5401 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     5461 ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca
     5521 attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     5581 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     5641 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     5701 agtttggtta attaaggtac cgagggccta tttcccatga ttccttcata tttgcatata
     5761 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     5821 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     5881 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     5941 ttatatatct tgtggaaagg acgaaacacc gcctgcaaca gatctttgat ggttttagag
     6001 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     6061 tcggtgcttt tttgaattcg ctagctaggt cttgaaagga gtgggaattg gctccggtgc
     6121 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     6181 caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     6241 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     6301 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gaccggttct agagcgctgc
     6361 caccatggac aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc
     6421 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     6481 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc
     6541 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
     6601 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
     6661 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
     6721 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     6781 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     6841 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     6901 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     6961 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     7021 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     7081 aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     7141 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     7201 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     7261 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     7321 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     7381 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     7441 cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     7501 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     7561 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     7621 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     7681 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     7741 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     7801 ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga
     7861 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     7921 cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     7981 cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt
     8041 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     8101 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     8161 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     8221 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     8281 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     8341 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     8401 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     8461 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     8521 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     8581 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     8641 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     8701 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     8761 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     8821 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     8881 ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag
     8941 aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     9001 gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa
     9061 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     9121 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     9181 gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa
     9241 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     9301 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     9361 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     9421 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     9481 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     9541 gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga
     9601 ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga
     9661 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct
     9721 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
     9781 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     9841 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     9901 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     9961 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
    10021 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
    10081 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
    10141 ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag
    10201 agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga
    10261 taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
    10321 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
    10381 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
    10441 gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga aggctggaca
    10501 ggctaagaag aagaaagatt acaaagacga tgacgataag ggatccggcg caacaaactt
    10561 ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc
    10621 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
    10681 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
    10741 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
    10801 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
    10861 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
    10921 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
    10981 caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
    11041 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
    11101 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
    11161 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa
    11221 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
    11281 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
    11341 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
    11401 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
    11461 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
    11521 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
    11581 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
    11641 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
    11701 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
    11761 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
    11821 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
    11881 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
    11941 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
    12001 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
    12061 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt
    12121 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
    12181 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
    12241 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
    12301 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
    12361 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
    12421 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
    12481 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
    12541 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
    12601 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
    12661 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
    12721 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
    12781 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
    12841 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
    12901 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
    12961 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac cat
//
