LOCUS       sequence-291972-        8512 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for SpyCas9 and puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8512)
  AUTHORS   Danner E, Lebedin M, de la Rosa K, Kuhn R
  TITLE     A homology independent sequence replacement strategy in human cells 
            using a CRISPR nuclease.
  JOURNAL   Open Biol. 2021 Jan;11(1):200283. doi: 10.1098/rsob.200283. Epub 
            2021 Jan 27.
  PUBMED    33499763
REFERENCE   2  (bases 1 to 8512)
  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: "Open 
            Biol."; date: "2021-01"; volume: "11(1):200283. doi"; pages: " 
            10.1098/rsob.200283. Epub 2021 Jan 27"
COMMENT     Sequence Label: pCbh-SpyCas9-2A-puro-pA_U6-BbsI
FEATURES             Location/Qualifiers
     source          1..8512
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        2..287
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        289..566
                     /label=chicken beta-actin promoter
     intron          567..795
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      807..816
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             816..881
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             888..908
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             933..5033
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             933..5033
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5034..5081
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5097..5150
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5151..5750
                     /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(5151..5170)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5647..5667
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5778..5795)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5784..5991
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6000..6129
                     /label=AAV2 ITR
     primer_bind     complement(6203..6221)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6289..6393
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6394..6462,6463..7254)
                     /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(6612..6631)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7425..8013
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7914..7933
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8075..8323
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8075..8095
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8246..8265
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8342..8417
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
       61 tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      121 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      181 acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc ttatgggact
      241 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
      301 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
      361 ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag
      421 gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
      481 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
      541 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc
      601 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
      661 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg
      721 tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa
      781 atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact
      841 acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc
      901 ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca
      961 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     1021 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     1081 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     1141 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     1201 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     1261 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     1321 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     1381 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     1441 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     1501 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     1561 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     1621 gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca
     1681 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     1741 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     1801 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     1861 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     1921 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     1981 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     2041 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     2101 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     2161 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     2221 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     2281 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     2341 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     2401 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     2461 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     2521 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     2581 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     2641 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     2701 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     2761 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     2821 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     2881 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     2941 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     3001 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     3061 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     3121 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     3181 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     3241 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     3301 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     3361 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     3421 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact
     3481 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     3541 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     3601 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     3661 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     3721 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     3781 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     3841 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     3901 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     3961 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     4021 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     4081 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     4141 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     4201 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     4261 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     4321 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     4381 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     4441 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     4501 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     4561 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     4621 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     4681 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     4741 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     4801 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     4861 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     4921 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     4981 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacaaaaggc
     5041 cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa ggaattcggc agtggagagg
     5101 gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca atgaccgagt
     5161 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     5221 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     5281 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     5341 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     5401 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     5461 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     5521 tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     5581 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     5641 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     5701 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga gaattctaac
     5761 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     5821 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     5881 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     5941 gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca
     6001 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     6061 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     6121 agcgcgcagc tgcctgcaat cactacattc acatctgatt tcagtagttc cttacgagag
     6181 ttcctccgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     6241 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     6301 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6361 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6421 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6481 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6541 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6601 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6661 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6721 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6781 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6841 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6901 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6961 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     7021 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     7081 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     7141 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     7201 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     7261 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     7321 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7381 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7441 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7501 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7561 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7621 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7681 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7741 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7801 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7861 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7921 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7981 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     8041 ttcctggcct tttgctggcc ttttgctcac atgtgagggc ctatttccca tgattccttc
     8101 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     8161 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     8221 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     8281 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgggtctt cgagaagacc
     8341 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     8401 tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta
     8461 gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagaggt ac
//
