LOCUS       sequence-362624-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets ESR1 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_ESR1
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..19
                     /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     8..30
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     27..43
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     27..43
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     62..82
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        64..82
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        110..336
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             337..517
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    564..689
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1182..1415
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1600..1644
                     /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    1825..1942
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2131..2342
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2361..2370
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2367..6470
                     /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             2367..6470
                     /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             6471..6518
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6519..6542
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6552..6608
                     /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             6609..7208
                     /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(6609..6628)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7105..7125
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7224..7812
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7277..7297)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7695..7706)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7968..8216
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7968..7988
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8139..8158
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8238..8313
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8366..8546
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8624..8745
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8661..8680)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8715..8734
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8785..8920
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8940..8959)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8941..8959)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8969..8986)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8969..8985)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8978..9000)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9127..9582
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9214..9233)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9424..9445
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9608..9712
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9713..9781,9782..10573)
                     /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(9931..9950)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10744..11332
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11233..11252
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11486..11503
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11620..11641
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11656..11661,11662..11679,11680..11686)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
       61 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat
      121 gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga
      181 gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta
      241 ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga
      301 gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc
      361 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      421 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      481 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      541 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      601 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      661 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      721 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      781 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      841 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      901 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      961 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     1021 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1081 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1141 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1201 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1261 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1321 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1381 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1441 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1501 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1561 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1621 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1681 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1741 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gaatcttgag
     1801 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
     1861 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     1921 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg
     1981 ccggctcgag ggggcccggg tgcaaagatg gataaagttt taaacagaga ggaatctttg
     2041 cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccgtag gtcttgaaag
     2101 gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     2161 agaagttggg gggaggggtc ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa
     2221 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     2281 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     2341 aggaccggtt ctagagcgct gccaccatgg acaagaagta cagcatcggc ctggacatcg
     2401 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     2461 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     2521 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     2581 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     2641 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     2701 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     2761 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     2821 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     2881 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     2941 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     3001 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     3061 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     3121 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     3181 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     3241 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     3301 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     3361 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     3421 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     3481 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     3541 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     3601 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     3661 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     3721 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     3781 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     3841 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     3901 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     3961 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     4021 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     4081 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     4141 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     4201 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     4261 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     4321 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     4381 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     4441 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     4501 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     4561 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     4621 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     4681 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     4741 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     4801 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     4861 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     4921 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     4981 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     5041 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     5101 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     5161 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     5221 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     5281 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     5341 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     5401 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     5461 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     5521 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     5581 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     5641 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     5701 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     5761 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     5821 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     5881 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     5941 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     6001 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     6061 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     6121 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     6181 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     6241 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     6301 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     6361 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     6421 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aagcgacctg
     6481 ccgccacaaa gaaggctgga caggctaaga agaagaaaga ttacaaagac gatgacgata
     6541 agggatccgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc
     6601 ctggaccgat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     6661 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     6721 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     6781 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca
     6841 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     6901 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     6961 ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg
     7021 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     7081 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     7141 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     7201 gtgcctgaac gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     7261 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     7321 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     7381 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     7441 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     7501 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     7561 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     7621 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     7681 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     7741 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     7801 ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca
     7861 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagattt
     7921 caccattatc gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc
     7981 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta
     8041 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     8101 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     8161 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggca
     8221 ccattgataa aaacagggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     8281 tatcaacttg aaaaagtggc accgagtcgg tgctttttta agcttggcgt aactagatct
     8341 tgagacactg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     8401 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     8461 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     8521 ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca
     8581 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     8641 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     8701 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     8761 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     8821 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     8881 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     8941 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     9001 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     9061 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     9121 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     9181 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     9241 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     9301 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     9361 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     9421 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     9481 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     9541 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     9601 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     9661 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     9721 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     9781 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     9841 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     9901 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     9961 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
    10021 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
    10081 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
    10141 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
    10201 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
    10261 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
    10321 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
    10381 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
    10441 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
    10501 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
    10561 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
    10621 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
    10681 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
    10741 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
    10801 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
    10861 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
    10921 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
    10981 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
    11041 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
    11101 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
    11161 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
    11221 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
    11281 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
    11341 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
    11401 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
    11461 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
    11521 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
    11581 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
    11641 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg g
//
