LOCUS       sequence-367170-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets MTOR gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11691)
  AUTHORS   Hu M, Lei XY, Larson JD, McAlonis M, Ford K, McDonald D, Mach K, 
            Rusert JM, Wechsler-Reya RJ, Mali P
  TITLE     Integrated genome and tissue engineering enables screening of cancer
            vulnerabilities in physiologically relevant perfusable ex vivo 
            cultures.
  JOURNAL   Biomaterials. 2022 Jan;280:121276. doi: 
            10.1016/j.biomaterials.2021.121276. Epub 2021 Dec 2.
  PUBMED    34890975
REFERENCE   2  (bases 1 to 11691)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Biomaterials."; date: "2022-01"; volume: "280:121276. doi"; pages: 
            " 10.1016/j.biomaterials.2021.121276. Epub 2021 Dec 2"
COMMENT     Sequence Label: CROP_C9_Puro_MTOR
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..23
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7..23
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     42..62
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        44..62
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        90..316
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             317..497
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    544..669
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1162..1395
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1580..1624
                     /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    1805..1922
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2111..2322
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2341..2350
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2347..6450
                     /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             2347..6450
                     /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             6451..6498
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6499..6522
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6532..6588
                     /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             6589..7188
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6589..6608)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7085..7105
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7204..7792
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7257..7277)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7675..7686)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7948..8196
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7948..7968
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8119..8138
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8218..8293
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8346..8526
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8604..8725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8641..8660)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8695..8714
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8765..8900
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8920..8939)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8921..8939)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8949..8966)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8949..8965)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8958..8980)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9107..9562
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9194..9213)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9404..9425
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9588..9692
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9693..9761,9762..10553)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(9911..9930)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10724..11312
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11213..11232
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11466..11483
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11600..11621
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11636..11641,11642..11659,11660..11666)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11674..11690
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     join(11679..11691,1..10)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
       61 gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg
      121 caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg
      181 ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct
      241 gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct
      301 agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
      361 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
      421 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
      481 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag
      541 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
      601 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
      661 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
      721 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
      781 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
      841 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
      901 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
      961 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt
     1021 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
     1081 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
     1141 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
     1201 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
     1261 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
     1321 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
     1381 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
     1441 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg
     1501 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga
     1561 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag
     1621 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat
     1681 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt
     1741 taggcaggga tattcaccat tatcgtttca gaatcttgag acaaatggca gtattcatcc
     1801 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     1861 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     1921 ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag ggggcccggg
     1981 tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg
     2041 tcttgaaagg agtgggaatt ggctccgtag gtcttgaaag gagtgggaat tggctccggt
     2101 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     2161 ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     2221 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     2281 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagagcgct
     2341 gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     2401 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     2461 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     2521 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     2581 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     2641 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     2701 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     2761 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     2821 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     2881 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     2941 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     3001 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     3061 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     3121 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     3181 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     3241 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     3301 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     3361 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     3421 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     3481 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     3541 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     3601 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     3661 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     3721 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     3781 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     3841 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     3901 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     3961 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     4021 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     4081 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     4141 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     4201 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     4261 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     4321 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     4381 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     4441 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     4501 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     4561 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     4621 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     4681 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     4741 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     4801 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     4861 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     4921 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     4981 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     5041 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     5101 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     5161 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     5221 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     5281 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     5341 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     5401 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     5461 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     5521 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     5581 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     5641 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     5701 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     5761 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     5821 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     5881 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     5941 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     6001 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     6061 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     6121 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     6181 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     6241 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     6301 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     6361 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     6421 cggatcgacc tgtctcagct gggaggcgac aagcgacctg ccgccacaaa gaaggctgga
     6481 caggctaaga agaagaaaga ttacaaagac gatgacgata agggatccgg cgcaacaaac
     6541 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgat gaccgagtac
     6601 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     6661 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     6721 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     6781 tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca cgccggagag cgtcgaagcg
     6841 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     6901 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     6961 ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     7021 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     7081 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     7141 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc
     7201 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     7261 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     7321 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     7381 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     7441 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     7501 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     7561 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     7621 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     7681 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     7741 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     7801 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     7861 gactggaagg gctaattcac tcccaacgaa gacaagattt caccattatc gtttcagacc
     7921 cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc ttcatatttg
     7981 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     8041 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     8101 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     8161 ttggctgtat atatcttgtg gaaaggacga aacaccgaat agggtgaatg atccggggtt
     8221 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     8281 accgagtcgg tgctttttta agcttggcgt aactagatct tgagacactg ctttttgctt
     8341 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     8401 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     8461 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     8521 ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     8581 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa
     8641 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     8701 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc
     8761 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     8821 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     8881 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac
     8941 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     9001 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     9061 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     9121 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     9181 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     9241 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     9301 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     9361 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     9421 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     9481 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     9541 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     9601 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     9661 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     9721 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     9781 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     9841 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     9901 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     9961 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
    10021 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
    10081 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
    10141 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
    10201 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
    10261 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
    10321 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
    10381 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
    10441 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
    10501 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
    10561 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
    10621 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
    10681 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
    10741 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
    10801 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
    10861 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
    10921 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
    10981 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
    11041 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
    11101 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
    11161 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
    11221 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
    11281 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
    11341 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
    11401 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
    11461 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
    11521 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
    11581 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
    11641 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa t
//
