pAAVS1-PDi-CRISPRn

LOCUS       pAAVS1-PDi-CRISPRn               12658 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Dox-inducible CRISPR nuclease (CRISPRn) knock in construct into the 
            AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    pAAVS1-PDi-CRISPRn
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12658)
  AUTHORS   Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan
            AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen 
            TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP, 
            Weissman JS, Qi LS, So PL, Conklin BR
  TITLE     CRISPR Interference Efficiently Induces Specific and Reversible Gene
            Silencing in Human iPSCs.
  JOURNAL   Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi: 
            10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.
  PUBMED    26971820
REFERENCE   2  (bases 1 to 12658)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..12658
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             54..914
                     /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(272..291)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1085..1673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1574..1593
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    1721..2557
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    2590..2608
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2626..2644
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2662..2680
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2698..2716
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2734..2752
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2770..2788
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2806..2824
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2869..2893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2977..2986
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2986..3051
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3058..3078
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3103..7203
                     /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             7204..7251
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    7286..7407
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7323..7342)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7377..7396
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7456..7472)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7456..7472)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7469..7491)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7480..7496
                     /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(7504..7534)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7549..7570
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7687..7704)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    complement(7760..7793)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     8124..8143
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    8142..8197
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     8243..8262
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(8352..9098)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(9130..9149)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          9157..10174
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(9211..9233)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(10176..10453)
                     /label=chicken beta-actin promoter
     enhancer        10455..10834
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    10840..11064
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     11053..11070
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(11102..11701)
                     /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(11185..11205)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     11682..11701
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(11711..11764)
                     /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"
     misc_feature    11788..11813
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    complement(11820..11853)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    11855..12658
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
ORIGIN
        1 aagggcgaat tcaaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
       61 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      241 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      301 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
      361 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
      421 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
      481 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
      541 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
      601 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
      661 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
      721 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
      781 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
      841 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
      901 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
      961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1021 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1201 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     1261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     1381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     1441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     1501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     1561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     1621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     1681 gcaacgcggc ctttttacgg ttcctggcga attcgccctt agagcagagc caggaacccc
     1741 tgtagggaag gggcaggaga gccaggggca tgagatggtg gacgaggaag ggggacaggg
     1801 aagcctgagc gcctctcctg ggcttgccaa ggactcaaac ccagaagccc agagcagggc
     1861 cttagggaag cgggaccctg ctctgggcgg aggaatatgt cccagatagc actggggact
     1921 ctttaaggaa agaaggatgg agaaagagaa agggagtaga ggcggccacg acctggtgaa
     1981 cacctaggac gcaccattct cacaaaggga gttttccaca cggacacccc cctcctcacc
     2041 acagccctgc caggacgggg ctggctactg gccttatctc acaggtaaaa ctgacgcacg
     2101 gaggaacaat ataaattggg gactagaaag gtgaagagcc aaagttagaa ctcaggacca
     2161 acttattctg attttgtttt tccaaactgc ttctcctctt gggaagtgta aggaagctgc
     2221 agcaccagga tcagtgaaac gcaccagaca gccgcgtcag agcagctcag gttctgggag
     2281 agggtagcgc agggtggcca ctgagaaccg ggcaggtcac gcatcccccc cttccctccc
     2341 accccctgcc aagctctccc tcccaggatc ctctctggct ccatcgtaag caaaccttag
     2401 aggttctggc aaggagagag atggctccag gaaatggggg tgtgtcacca gataaggaat
     2461 ctgcctaaca ggaggtgggg gttagaccca atatcaggag actaggaagg aggaggccta
     2521 aggatggggc ttttctgtca ccaatcctgt ccctagtaaa gctgatatct ccaccccaca
     2581 gtggtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
     2641 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
     2701 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
     2761 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
     2821 gagaacgtat aagctttagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt
     2881 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta
     2941 ccacttccta ccctcgtaaa cttaaggtta attaacgcca ccatggacta taaggaccac
     3001 gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca
     3061 aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc
     3121 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     3181 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     3241 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
     3301 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     3361 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     3421 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     3481 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     3541 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     3601 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     3661 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     3721 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     3781 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
     3841 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     3901 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     3961 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     4021 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     4081 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     4141 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     4201 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     4261 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     4321 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     4381 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     4441 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     4501 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
     4561 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     4621 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     4681 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
     4741 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     4801 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     4861 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     4921 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     4981 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     5041 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     5101 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     5161 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     5221 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     5281 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     5341 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     5401 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     5461 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     5521 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     5581 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
     5641 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
     5701 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     5761 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     5821 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     5881 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     5941 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     6001 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     6061 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     6121 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     6181 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     6241 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     6301 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     6361 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     6421 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     6481 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     6541 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     6601 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     6661 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     6721 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     6781 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     6841 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     6901 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     6961 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     7021 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     7081 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     7141 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     7201 gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaaaccggt
     7261 gcggccgcaa tcgatcgccc accccaactt gtttattgca gcttataatg gttacaaata
     7321 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     7381 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     7441 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     7501 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     7561 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     7621 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     7681 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     7741 tcactcaaag gacggccgca taacttcgta taatgtatgc tatacgaagt tataagcttg
     7801 ggctgcaggt cgagggatct ccataagaga agagggacag ctatgactgg gagtagtcag
     7861 gagaggagga aaaatctggc tagtaaaaca tgtaaggaaa attttaggga tgttaaagaa
     7921 aaaaataaca caaaacaaaa tataaaaaaa atctaacctc aagtcaaggc ttttctatgg
     7981 aataaggaat ggacagcagg gggctgtttc atatactgat gacctcttta tagccaacct
     8041 ttgttcatgg cagccagcat atgggcatat gttgccaaac tctaaaccaa atactcattc
     8101 tgatgtttta aatgatttgc cctcccatat gtccttccga gtgagagaca caaaaaattc
     8161 caacacacta ttgcaatgaa aataaatttc ctttattagc cagaagtcag atgctcaagg
     8221 ggcttcatga tgtccccata atttttggca gagggaaaaa gatctcagtg gtatttgtga
     8281 gccagggcat tggccacacc agccaccacc ttctgatagg cagcctgcac ctgaggagtg
     8341 aattcacgcg tttagcctgg cagcatatcg agatcgaagt catcgagagc gtcagcaggg
     8401 agcatatcca gatcaaaatc atccagggca tcggcgggca gcatgtccag gtcgaagtcg
     8461 tccagggcgt cggtgggtcc gccgctctcg cacttcagct gcttctcgag gccgcagatg
     8521 atcagttcca ggccgaacag gaaggcgggc tcggcgccct gtctgtcgaa cagctcgatg
     8581 gcctgcttca gcaggggggg catgctgtcg gtggtgggtg tctctctttc ctctttggcg
     8641 acctggtgtt cctgttcttc cagcacgcag cccagggtga agtggcccac ggcgctcagg
     8701 gcgtacaggg cgttttccag gctgaagccc tgctggcaca ggaaggccag ctggttttcc
     8761 agtgtctcgt actgcttctc ggtgggtctg gtgcccaggt gcactttggc gccgtcccgg
     8821 tggctcagca gggcgcatct gtagctcttg gcgttgttcc gcaggaagtc ctgccagctc
     8881 tcgccttcca gagggcagct gtgggtgtgg tgccggtcca gcatctcgat gggcagggcg
     8941 tccagcaggg cccgcttgtt cttcacgtgc cagtacaggg tgggctgttc cacgcccagt
     9001 ttctgggcca gcttccgggt ggtcaggccc tcgatgccca cgccgttcag cagttccagg
     9061 gcgctgttga tcactttgct cttgtccagc cggctcatgt cgacattctt tgccaaaatg
     9121 atgagacagc acaataacca gcacgttgcc caggagctgt aggaaaaaga agaaggcatg
     9181 aacatggtta gcagaggctc tagagccgcc ggtcacacgc cagaagccga accccgccct
     9241 gccccgtccc ccccgaaggc agccgtcccc ccgcggacag ccccgaggct ggagagggag
     9301 aaggggacgg cggcgcggcg acgcacgaag gccctccccg cccatttcct tcctgccggc
     9361 gccgcaccgc ttcgccccgc gcccgctaga gggggtgcgg cggcgcctcc cagatttcgg
     9421 ctccgcacag atttgggaca aaggaagtcc ctgcgccctc tcgcacgatt accataaaag
     9481 gcaatggctg cggctcgccg cgcctcgaca gccgccggcg ctccgggggc cgccgcgccc
     9541 ctcccccgag ccctccccgg cccgaggcgg ccccgccccg cccggcaccc ccacctgccg
     9601 ccaccccccg cccggcacgg cgagccccgc gccacgcccc gtacggagcc ccgcacccga
     9661 agccgggccg tgctcagcaa ctcggggagg ggggtgcagg gggggttgca gcccgaccga
     9721 cgcgcccaca ccccctgctc acccccccac gcacacaccc cgcacgcagc ctttgttccc
     9781 ctcgcagccc cccccgcacc gcggggcacc gcccccggcc gcgctcccct cgcgcacact
     9841 gcggagcgca caaagccccg cgccgcgccc gcagcgctca cagccgccgg gcagcgcgga
     9901 gccgcacgcg gcgctcccca cgcacacaca cacgcacgca ccccccgagc cgctcccccc
     9961 gcacaaaggg ccctcccgga gcccctcaag gctttcacgc agccacagaa aagaaacaag
    10021 ccgtcattaa accaagcgct aattacagcc cggaggagaa gggccgtccc gcccgctcac
    10081 ctgtgggagt aacgcggtca gtcagagccg gggcgggcgg cgcgaggcgg cggcggagcg
    10141 gggcacgggg cgaaggcagc gtcgcagcga ctccccgccc gccgcgcgct tcgcttttta
    10201 tagggccgcc gccgccgccg cctcgccata aaaggaaact ttcggagcgc gccgctctga
    10261 ttggctgccg ccgcacctct ccgcctcgcc ccgccccgcc cctcgccccg ccccgccccg
    10321 cctggcgcgc gccccccccc cccccccgcc cccatcgctg cacaaaataa ttaaaaaata
    10381 aataaataca aaattggggg tggggagggg ggggagatgg ggagagtgaa gcagaacgtg
    10441 gggctcacct cgaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
    10501 aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
    10561 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
    10621 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
    10681 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
    10741 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
    10801 gattactatt aataactagt caataatcaa tgtcgagacc catagagccc accgcatccc
    10861 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
    10921 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
    10981 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
    11041 atggctggca actagaaggc acagtcgagg ctgatcagcg ggtttaaacg ggccctctag
    11101 atcaggcacc gggcttgcgg gtcatgcacc aggtgcgcgg tccttcgggc acctcgacgt
    11161 cggcggtgac ggtgaagccg agccgctcgt agaaggggag gttgcggggc gcggaggtct
    11221 ccaggaaggc gggcaccccg gcgcgctcgg ccgcctccac tccggggagc acgacggcgc
    11281 tgcccagacc cttgccctgg tggtcgggcg agacgccgac ggtggccagg aaccacgcgg
    11341 gctccttggg ccggtgcggc gccaggaggc cttccatctg ttgctgcgcg gccagccggg
    11401 aaccgctcaa ctcggccatg cgcgggccga tctcggcgaa caccgccccc gcttcgacgc
    11461 tctccggcgt ggtccagacc gccaccgcgg cgccgtcgtc cgcgacccac accttgccga
    11521 tgtcgagccc gacgcgcgtg aggaagagtt cttgcagctc ggtgacccgc tcgatgtggc
    11581 ggtccggatc gacggtgtgg cgcgtggcgg ggtagtcggc gaacgcggcg gcgagggtgc
    11641 gtacggccct ggggacgtcg tcgcgggtgg cgaggcgcac cgtgggcttg tactcggtca
    11701 tctcgagcct agggccggga ttctcctcca cgtcaccgca tgttagaaga cttcctctgc
    11761 cctctccgct gccagatctc tcgaggccct gtgggaggaa gagaagaggt cagaagctta
    11821 taacttcgta taatgtatgc tatacgaagt tattgcccca ctgtggggtg gaggggacag
    11881 ataaaagtac ccagaaccag agccacatta accggccctg ggaatataag gtggtcccag
    11941 ctcggggaca caggatccct ggaggcagca aacatgctgt cctgaagtgg acataggggc
    12001 ccgggttgga ggaagaagac tagctgagct ctcggacccc tggaagatgc catgacaggg
    12061 ggctggaaga gctagcacag actagagagg taaggggggt aggggagctg cccaaatgaa
    12121 aggagtgaga ggtgacccga atccacagga gaacggggtg tccaggcaaa gaaagcaaga
    12181 ggatggagag gtggctaaag ccagggagac ggggtacttt ggggttgtcc agaaaaacgg
    12241 tgatgatgca ggcctacaag aaggggaggc gggacgcaag ggagacatcc gtcggagaag
    12301 gccatcctaa gaaacgagag atggcacagg ccccagaagg agaaggaaaa gggaacccag
    12361 cgagtgaaga cggcatgggg ttgggtgagg gaggagagat gcccggagag gacccagaca
    12421 cggggaggat ccgctcagag gacatcacgt ggtgcagcgc cgagaaggaa gtgctccgga
    12481 aagagcatcc ttgggcagca acacagcaga gagcaagggg aagagggagt ggaggaagac
    12541 ggaacctgaa ggaggcggca gggaaggatc tgggccagcc gtagaggtga cccaggccac
    12601 aagctgcaga cagaaagcgg cacaggccca ggggagagaa tgctggtcag agaaagca
//