pCRISPR-aBEST

LOCUS       pCRISPR-aBEST               12449 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of a Streptomyces codon optimized Cas9n-tadA fusion 
            protein in pGM1190. A to G base editor for actinomycetes.
ACCESSION   .
VERSION     .
KEYWORDS    pCRISPR-aBEST
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12449)
  AUTHORS   Tong Y, Whitford CM, Robertsen HL, Blin K, Jorgensen TS, Klitgaard 
            AK, Gren T, Jiang X, Weber T, Lee SY
  TITLE     Highly efficient DSB-free base editing for streptomycetes with 
            CRISPR-BEST.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Oct 8;116(41):20366-20375. doi: 
            10.1073/pnas.1913493116. Epub 2019 Sep 23.
  PUBMED    31548381
REFERENCE   2  (bases 1 to 12449)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..12449
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     oriT            466..575
                     /note="incP origin of transfer"
     CDS             891..1691
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDSFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELLAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     rep_origin      1993..2580
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2481..2500
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2734..2751
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2868..2889
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2904..2934
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2942..2958
                     /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     2947..2969
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2966..2982
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2966..2982
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        3120..3195
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3365..3399
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             4758..8861
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     terminator      9048..9096
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             10811..12253
                     /codon_start=1
                     /gene="rep"
                     /product="replication initiator protein from the 
                     Streptomyces ghanaensis plasmid pSG5"
                     /label=pSG5 Rep
                     /translation="MWKPGEATWGNTCRCNNVHTCPWCMSRILAVRGSNVQLAADGLAD
                     AGYGLHLGTNTLRHFERMAFGTVRKGMRHGLVAVLHDGWKGAYGSSGRRWRTMRDDFGI
                     IGYERAFEDTFGWGSGWHLHWHTLWVTREVLGPDAQAAFRDALAGAWAAGVESAGGYTV
                     SETCDRPGCSCEGKGHGTDVRPLNGADAADGDAGKQARYLYKDGDKTKGGVAKIGLELA
                     GQNFKAGRGDDRMGPLDLGDAAAAELQRLRRPGPFVEKYREREFGVFQVRKHYRSQNLN
                     RLIKELGIQQDVRTEEEITDDTEGLVAIAVIPAYIWYRYIARVAGRRLDLIKVAETYGL
                     PGVRRLVESWGLVWGKDVLDPPAPEAPAAPGDLDADQMRFEVMSEEEAAFREARRKANE
                     ARTEELAASLDRVRQPKKEAIRPTISLRKRLKPKPVTVDVKTPPPGAASPVCRRCKGKL
                     APVLQPWGQHPGDCLRVDTAVA"
ORIGIN
        1 ggccgtcgac gacgacgcgt ccgcctgcct gcttttggcc ggcgggccgg gccgcggttg
       61 tccgccgggt cggaccggtc ccttggtcga ccgatgcctc ggccacgacg atgctgggcc
      121 cgaagcggta cgcgacccgt gccgggccgc agcggatcgc ggcagcagga gctccccgcc
      181 cgccacggct ggctggatgg tcgcctcacg cgcccgcagg ggtgacggcg tgggtgcggc
      241 ggtcagcccc gtgatcgggc gtcagacggc cgctgaggga ccgcgaccac ccctcccttc
      301 ccgcctgccg tctttcaccg cccggccggt ccgcagacgc cgtacggcca ggccacggag
      361 gattacgggc agatggcgcc cacccggcac ccgggctggg gcaccctgga cagcacgaag
      421 gtccgcccca ctcaccgggg cgggccttcg cgttgagcta gagttcccgc cagcctcgca
      481 gagcaggatt cccgttgagc accgccaggt gcgaataagg gacagtgaag aaggaacacc
      541 cgctcgcggg tgggcctact tcacctatcc tgcccggctg acgccgttgg atacaccaag
      601 gaaagtctac acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata
      661 ccgaaaaaat cgctataatg accccgaagc agggttatgc agcggaaaat gcagctcacg
      721 gtaactgatg ccgtatttgc agtaccagcg tacggcccac agaatgatgt cacgctgaaa
      781 atgccggcct ttgaatgggt tcatgtgcag ctccataagc aaaaggggat gataagttta
      841 tcaccaccga ctatttgcaa cagtgccgtt gatcgtgcta tgatcgactg atgtcatcag
      901 cggtggagtg caatgtcgtg caatacgaat ggcgaaaagc cgagctcatc ggtcagcttc
      961 tcaaccttgg ggttaccccc ggcggtgtgc tgctggtcca cagctccttc cgtagcgtcc
     1021 ggcccctcga agatgggcca cttggactga tcgaggccct gcgtgctgcg ctgggtccgg
     1081 gagggacgct cgtcatgccc tcgtggtcag gtctggacga cgagccgttc gatcctgcca
     1141 cgtcgcccgt tacaccggac cttggagttg tctctgactc attctggcgc ctgccaaatg
     1201 taaagcgcag cgcccatcca tttgcctttg cggcagcggg gccacaggca gagcagatca
     1261 tctctgatcc attgcccctg ccacctcact cgcctgcaag cccggtcgcc cgtgtccatg
     1321 aactcgatgg gcaggtactt ctcctcggcg tgggacacga tgccaacacg acgctgcatc
     1381 ttgccgagtt gttggcaaag gttccctatg gggtgccgag acactgcacc attcttcagg
     1441 atggcaagtt ggtacgcgtc gattatctcg agaatgacca ctgctgtgag cgctttgcct
     1501 tggcggacag gtggctcaag gagaagagcc ttcagaagga aggtccagtc ggtcatgcct
     1561 ttgctcggtt gatccgctcc cgcgacattg tggcgacagc cctgggtcaa ctgggccgag
     1621 atccgttgat cttcctgcat ccgccagagg cgggatgcga agaatgcgat gccgctcgcc
     1681 agtcgattgg ctgagctcat aagttcctat tccgaagttc ctattcgaaa tgaccgacca
     1741 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     1801 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     1861 gctggagttc ttcgcccacc ccaaaaggat ctaggtgaag atcctttttg ataatctcat
     1921 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1981 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2041 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2101 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2161 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2221 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2281 gttaccggat aaggcgcagc ggtcgggctg aacggggggt cgtgcacaca gcccagcttg
     2341 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2401 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     2461 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     2521 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     2581 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     2641 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     2701 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     2761 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     2821 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     2881 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     2941 attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac gaattgtacg
     3001 cggtcgatct tgacggctgg cgagaggtgc ggggaggatc tgaccgacgc ggtccacacg
     3061 tggcaccgcg atgctgttgt gggcacaatc gtgccggttg gtaggatcga cggccatggg
     3121 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     3181 gcaccgagtc ggtgcttttt ttacgtaggc gtcagctacg atgttccggg gactgctgat
     3241 ccggtcagca ggtggaagag ggactggatt ccaaagttct caatgctgct tgctgttctt
     3301 gaatgggggg tcgttgacga cgacatggct cgattggcgc gacaagttgc tgcgattctc
     3361 accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga
     3421 ggtcattact ggaccggatc ggggatctgg gctgagggag ccgacggcac gcggcggctc
     3481 acggcgtggc acgcggaacg tccgggcttg cacctcacgt cacgtgagga ggcagcgtgg
     3541 acggcgtcag agaagggagc ggacatatga gcgaggtcga gttctcccac gagtactgga
     3601 tgcggcacgc cctgaccctg gcgaagcggg cctgggacga gcgcgaggtc cccgtcggcg
     3661 ccgtcctggt ccacaacaac cgggtcatcg gcgagggctg gaaccggccc atcggccggc
     3721 acgaccccac cgcccacgcc gagatcatgg ccctgcgcca gggcggcctg gtcatgcaga
     3781 actaccggct gatcgacgcc accctgtacg tcaccctgga gccctgcgtc atgtgcgccg
     3841 gcgccatgat ccactcccgc atcggccggg tcgtcttcgg cgcccgcgac gccaagaccg
     3901 gcgccgccgg ctccctgatg gacgtcctgc accaccccgg catgaaccac cgcgtcgaga
     3961 tcaccgaggg catcctggcc gacgagtgcg ccgccctgct gtccgacttc ttccggatgc
     4021 gccggcagga gatcaaggcc cagaagaagg cccagtcctc caccgactcc ggcggctcct
     4081 ccggcggctc ctccggctcc gagacgccgg gcacctcgga gtccgccacc ccggagtcgt
     4141 ccggcggctc ctccggcggc tcctccgagg tcgagttctc gcacgagtac tggatgcgcc
     4201 acgccctgac cctggccaag cgggcccggg acgaacgcga ggtcccggtg ggcgcggtcc
     4261 tggtcctgaa caaccgggtg attggtgagg gctggaaccg cgccatcggc ctgcacgacc
     4321 cgaccgcgca cgcggagatc atggcgctcc ggcagggcgg cctggtgatg cagaactacc
     4381 gcctcatcga cgcgaccctc tacgtcacct tcgagccctg cgtgatgtgc gcgggcgcga
     4441 tgattcactc ccgcattggc cgggtggtct tcggcgtccg caacgcgaag accggcgcgg
     4501 cgggctccct gatggacgtg ctgcactacc cgggcatgaa ccaccgggtg gaaattaccg
     4561 agggcatcct ggcggacgag tgcgcggcgc tgctgtgcta cttcttccgc atgccccggc
     4621 aggtcttcaa cgcgcagaag aaggcccaat cctccaccga ttccggcggc tcctccggcg
     4681 gctcctccgg ctccgaaacc ccgggcacct cggagtcggc caccccggag tccagcggcg
     4741 gcagcagcgg cggctcggac aagaagtact ccatcggcct cgcgatcggc accaactccg
     4801 tgggctgggc ggtcatcacc gacgagtaca aggtcccctc caagaagttc aaggtcctgg
     4861 gcaacaccga ccggcactcg atcaagaaga acctgatcgg cgccctgctc ttcgacagcg
     4921 gcgagaccgc cgaggcgacc cgcctgaagc ggaccgcgcg tcgccgctac acccggcgca
     4981 agaaccgcat ctgctacctg caggaaatct tctccaacga gatggccaag gtggacgact
     5041 cgttcttcca ccgcctggag gagagcttcc tggtggagga ggacaagaag cacgagcgcc
     5101 acccgatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     5161 accacctccg caagaagctg gtggactcga ccgacaaggc ggacctgcgg ctcatctacc
     5221 tggccctcgc gcacatgatc aagttccgcg gccacttcct catcgagggc gacctgaacc
     5281 cggacaactc cgacgtggac aagctcttca tccagctggt gcagacctac aaccagctgt
     5341 tcgaggagaa ccccatcaac gccagcggcg tggacgccaa ggcgatcctc tccgcgcgcc
     5401 tgagcaagtc ccggcgcctg gagaacctca tcgcccagct gccgggcgag aagaagaacg
     5461 gcctcttcgg caacctgatc gcgctgtcgc tcggcctgac ccccaacttc aagagcaact
     5521 tcgacctggc cgaggacgcg aagctccagc tgtccaagga cacctacgac gacgacctgg
     5581 acaacctgct cgcccagatc ggcgaccagt acgcggacct cttcctggcc gcgaagaacc
     5641 tctcggacgc catcctgctc agcgacatcc tgcgggtcaa caccgagatc accaaggccc
     5701 cgctgtcggc gagcatgatc aagcggtacg acgagcacca ccaggacctg accctgctca
     5761 aggccctcgt gcgccagcag ctgcccgaga agtacaagga aatcttcttc gaccagtcca
     5821 agaacggcta cgccggctac atcgacggcg gcgcgtcgca ggaggagttc tacaagttca
     5881 tcaagccgat cctggagaag atggacggca ccgaggagct gctcgtcaag ctgaaccgcg
     5941 aggacctgct ccgcaagcag cggaccttcg acaacggctc catcccgcac cagatccacc
     6001 tgggcgagct ccacgccatc ctccggcgcc aggaggactt ctaccccttc ctgaaggaca
     6061 accgcgagaa gatcgagaag atcctgacct tccgcatccc gtactacgtc ggccccctgg
     6121 cccgcggcaa ctcccggttc gcgtggatga cccggaagtc ggaggagacc atcaccccgt
     6181 ggaacttcga ggaggtcgtg gacaagggcg cgtccgcgca gtcgttcatc gagcgcatga
     6241 ccaacttcga caagaacctc ccgaacgaga aggtcctgcc caagcactcc ctgctctacg
     6301 agtacttcac cgtgtacaac gagctgacca aggtcaagta cgtgaccgag ggcatgcgga
     6361 agccggcctt cctgtcgggc gagcagaaga aggcgatcgt ggacctgctc ttcaagacca
     6421 accgcaaggt caccgtgaag cagctgaagg aggactactt caagaagatc gagtgcttcg
     6481 actccgtcga gatcagcggc gtggaggacc gcttcaacgc ctccctgggc acctaccacg
     6541 acctgctcaa gatcatcaag gacaaggact tcctcgacaa cgaggagaac gaggacatcc
     6601 tggaggacat cgtcctcacc ctgaccctct tcgaggaccg cgagatgatc gaggagcggc
     6661 tcaagaccta cgcccacctg ttcgacgaca aggtgatgaa gcagctgaag cgtcgccgct
     6721 acaccggctg gggccgcctc tcccggaagc tgatcaacgg catccgggac aagcagagcg
     6781 gcaagaccat cctggacttc ctcaagtccg acggcttcgc caaccgcaac ttcatgcagc
     6841 tcatccacga cgacagcctg accttcaagg aggacatcca gaaggcccag gtctcgggcc
     6901 agggcgacag cctccacgag cacatcgcca acctggcggg ctccccggcg atcaagaagg
     6961 gcatcctcca gaccgtcaag gtcgtggacg agctggtcaa ggtgatgggc cgccacaagc
     7021 ccgagaacat cgtgatcgag atggcccggg agaaccagac cacccagaag ggccagaaga
     7081 actcgcgcga gcggatgaag cggatcgagg agggcatcaa ggagctcggc agccagatcc
     7141 tgaaggagca cccggtcgag aacacccagc tgcagaacga gaagctgtac ctctactacc
     7201 tgcagaacgg ccgcgacatg tacgtggacc aggagctcga catcaaccgg ctgtccgact
     7261 acgacgtgga ccacatcgtg ccgcagtcct tcctgaagga cgactcgatc gacaacaagg
     7321 tcctgacccg ctcggacaag aaccggggca agtccgacaa cgtgccctcg gaggaggtcg
     7381 tgaagaagat gaagaactac tggcgccagc tgctcaacgc caagctcatc acccagcgca
     7441 agttcgacaa cctgaccaag gccgagcggg gcggcctgag cgagctcgac aaggcgggct
     7501 tcatcaagcg ccagctggtc gagacccggc agatcaccaa gcacgtggcc cagatcctgg
     7561 actcccggat gaacaccaag tacgacgaga acgacaagct gatccgcgag gtcaaggtga
     7621 tcaccctcaa gagcaagctg gtctccgact tccgcaagga cttccagttc tacaaggtcc
     7681 gggagatcaa caactaccac cacgcccacg acgcgtacct gaacgccgtc gtgggcaccg
     7741 cgctgatcaa gaagtacccg aagctggagt ccgagttcgt ctacggcgac tacaaggtct
     7801 acgacgtgcg caagatgatc gccaagagcg agcaggagat cggcaaggcc accgcgaagt
     7861 acttcttcta ctccaacatc atgaacttct tcaagaccga gatcaccctg gccaacggcg
     7921 agatccgcaa gcggcccctg atcgagacca acggcgagac cggcgagatc gtctgggaca
     7981 agggccgcga cttcgccacc gtccggaagg tgctgtcgat gccgcaggtc aacatcgtga
     8041 agaagaccga ggtgcagacc ggcggcttca gcaaggagtc catcctcccc aagcgcaaca
     8101 gcgacaagct gatcgcccgg aagaaggact gggacccgaa gaagtacggc ggcttcgaca
     8161 gccccaccgt cgcctactcc gtgctggtcg tggcgaaggt cgagaagggc aagagcaaga
     8221 agctgaagtc cgtgaaggag ctgctcggca tcaccatcat ggagcgctcc tcgttcgaga
     8281 agaacccgat cgacttcctg gaggccaagg gctacaagga ggtcaagaag gacctcatca
     8341 tcaagctgcc caagtacagc ctgttcgagc tggagaacgg ccgcaagcgg atgctcgcct
     8401 ccgcgggcga gctgcagaag ggcaacgagc tggccctccc gtcgaagtac gtcaacttcc
     8461 tgtacctcgc gtcccactac gagaagctga agggctcgcc cgaggacaac gagcagaagc
     8521 agctcttcgt ggagcagcac aagcactacc tggacgagat catcgagcag atcagcgagt
     8581 tcagcaagcg cgtcatcctg gccgacgcga acctcgacaa ggtgctgtcc gcctacaaca
     8641 agcaccgcga caagccgatc cgggagcagg cggagaacat catccacctg ttcaccctca
     8701 ccaacctggg cgcccccgcc gcgttcaagt acttcgacac caccatcgac cgcaagcggt
     8761 acacctccac caaggaggtc ctcgacgcga ccctgatcca ccagagcatc accggcctgt
     8821 acgagacccg catcgacctg tcccagctcg gcggcgactg aaagctttct agaggatccc
     8881 cgggtaccga gctcgaattc cccagatcta aagttttgtc gtctttccag acgttagtaa
     8941 atgaattttc tgtatgaggt tttgctaaac aactttcaac agtttcagcg gagtgagaat
     9001 agaaaggaac aactaaagga attgcgaata ataatttttt cacgttgaaa atctccaaaa
     9061 aaaaaggctc caaaaggagc ctttaattgt atcggtttat cagcttgctt tcgaggtgaa
     9121 tttcttaaac agcttgatac cgatagttgc gccgacaatg acaacaacca tcgcccacgc
     9181 ataaccgata tattcggtcg ctgaggcttg cagggagtca aaggccgctt ttgcgggatc
     9241 tcgtcgaagg cactagaggc ctatatgcgg ggatcgaccg cgcgggtccc ggacggggaa
     9301 gagcggggag ctttgccaga gagcgacgac ttccccttgc gttggtgatt gccggtcagg
     9361 gcagccatcc gccatcgtcg cgtagggtgt cacaccccag gaatcgcgtc actgaacaca
     9421 gcagccggta ggacgaccat gactgagttg gacaccatcg caaatccgtc cgatcccgcg
     9481 gtgcagcgga tcatcgatgt caccaagccg tcgcgatcca acataaagac aacgttgatc
     9541 gaggacgtcg agcccctcat gcacagcatc gcggccgggg tggagttcat cgaggtctac
     9601 ggcagcgaca gcagtccttt tccatctgag ttgctggatc tgtgcgggcg gcagaacata
     9661 ccggtccgcc tcatcgactc ctcgatcgtc aaccagttgt tcaaggggga gcggaaggcc
     9721 aagacattcg gcatcgcccg cgtccctcgc ccggccaggt tcggcgatat cgcgagccgg
     9781 cgtggggacg tcgtcgttct cgacggggtg aagatcgtcg ggaacatcgg cgcgatagta
     9841 cgcacgtcgc tcgcgctcgg agcgtcgggg atcatcctgg tcgacagtga catcaccagc
     9901 atcgcggacc ggcgtctcca aagggccagc cgaggttacg tcttctccct tcccgtcgtt
     9961 ctctccggtc gcgaggaggc catcgccttc attcgggaca gcggtatgca gctgatgacg
    10021 ctcaaggcgg atggcgacat ttccgtgaag gaactcgggg acaatccgga tcggctggcc
    10081 ttgctgttcg gcagcgaaaa gggtgggcct tccgacctgt tcgaggaggc gtcttccgcc
    10141 tcggtttcca tccccatgat gagccagacc gagtctctca acgtttccgt ttccctcgga
    10201 atcgcgctgc acgagaggat cgacaggaat ctcgcggcca accgataagc gcctctgttc
    10261 ctcggacgct cggttgctcg acctcgattc gtcagtgatg atcaacagga cccagaaatg
    10321 gcacgagccc gggattggcc tcccgggctc gccgcccccg aaccggcgca gcatgccgag
    10381 tgaggacgtt tgctctgtga cgagcgtacg agagaacagc gcgtcgacac caccgcgccc
    10441 cgccggaccg aagtgccact gcggcacccc gatcgtccac atccccggga agcgccccaa
    10501 aatctactgc tcggacgcct gccgaaagcg cgcaaagcgt ctcattgcgg accagtcacg
    10561 cagcgtagca agcacgacta cagcgggtaa tccggacgcg ggctttagta gtaagggaaa
    10621 atctccgtcc actgcaggtc agagcgctgt aactggaacg ggttctaccc caaacgaggg
    10681 tgccgcgaca gccgggcggg aggacccgcg cgacgagcgg ttcgcccggc gggaccggca
    10741 ccagacggtg tcgctgaacg aggcgttcaa gggctgcggg acccggctca ctgccgggac
    10801 ggccgagctg atgtggaagc cgggcgaggc gacgtggggc aacacgtgcc gctgcaacaa
    10861 cgtccacacc tgcccgtggt gcatgagccg catcctggcc gtacggggct cgaacgtgca
    10921 gctcgccgcc gacggtctcg cggacgccgg gtacgggctg caccttggca cgaacacgtt
    10981 gcgccacttc gagcgcatgg cgttcggcac cgtccgcaag ggaatgcggc acggcctggt
    11041 cgcggtcttg cacgacggct ggaagggcgc gtacggctcg tccggccgcc gctggcgcac
    11101 gatgcgcgac gacttcggga tcatcggcta cgagcgggcc ttcgaggaca ccttcggctg
    11161 gggctccggc tggcacctgc actggcacac gctctgggtc acccgcgagg tgctcggccc
    11221 ggacgcccag gccgcgttcc gcgacgcact cgccggagcc tgggccgccg gggtcgaatc
    11281 cgcgggcggc tacaccgtca gcgagacgtg cgaccggccc ggttgctcct gtgagggcaa
    11341 gggccacggc accgacgtgc gcccactcaa cggcgcggac gccgctgacg gcgatgcggg
    11401 caagcaggcc cgctacctct acaaggacgg cgacaagacc aagggcggcg tcgccaagat
    11461 cggcctggag ctggccggac agaacttcaa ggccggtcgc ggcgacgacc gcatgggacc
    11521 gctcgacctg ggcgacgcgg cggccgccga gctgcagcgg cttcgtcggc ccggcccgtt
    11581 cgtcgagaag taccgcgagc gcgagttcgg cgtcttccag gtccgcaagc actaccggtc
    11641 gcagaacctg aaccggctga tcaaggaact cggcatccag caggacgtcc gcaccgagga
    11701 ggagatcacc gacgacaccg agggcctggt cgccatcgcg gtcatcccgg cctacatctg
    11761 gtaccgatac atcgcccgcg tcgccggacg tcgcctcgac ctcatcaagg tcgccgagac
    11821 gtacggccta ccgggcgtcc ggcggctcgt cgagtcctgg ggcctggtgt ggggcaagga
    11881 cgtcctggac ccgcccgccc cggaagcgcc ggccgcgccg ggcgaccttg acgctgacca
    11941 gatgaggttc gaggtcatgt ccgaggagga agctgctttc cgcgaggcgc gccggaaggc
    12001 caacgaggcc cgcacggagg agctggccgc ttcgctcgac cgagtgcgtc agccgaagaa
    12061 ggaggcgatc cgcccgacga tcagccttcg gaagcgcctg aagccgaagc cggtcaccgt
    12121 cgacgtcaag actcccccgc ccggcgccgc gtcgcccgtg tgccgccgct gcaagggcaa
    12181 gctcgccccg gtactccagc cctggggcca gcaccccgga gactgcctcc gcgtcgacac
    12241 agcggtcgcg tagggccgct gagggaccgc catgagaccg ccaccccgga catccgggtg
    12301 gcggtctcat ggcggtcccg gcgcgaaagc cgagggtctg cctgccgtga ggtggccggc
    12361 ggacctgggt tactgctgga atcccgcgcc cagtccgagc gtccgccggc gcttttcacc
    12421 tggctccggg tgctcgtcga tgagcacca
//