pCRISPR-cBEST

LOCUS       pCRISPR-cBEST               12247 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  C to T base editor for actinomycetes.
ACCESSION   .
VERSION     .
KEYWORDS    pCRISPR-cBEST
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12247)
  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 12247)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..12247
                     /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        3119..3194
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3364..3398
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             3566..4252
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             4301..8401
                     /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"
     CDS             8414..8665
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     terminator      8846..8894
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             10609..12051
                     /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 cggccatggt
     3121 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     3181 caccgagtcg gtgctttttt tacgtaggcg tcagctacga tgttccgggg actgctgatc
     3241 cggtcagcag gtggaagagg gactggattc caaagttctc aatgctgctt gctgttcttg
     3301 aatggggggt cgttgacgac gacatggctc gattggcgcg acaagttgct gcgattctca
     3361 ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag
     3421 gtcattactg gaccggatcg gggatctggg ctgagggagc cgacggcacg cggcggctca
     3481 cggcgtggca cgcggaacgt ccgggcttgc acctcacgtc acgtgaggag gcagcgtgga
     3541 cggcgtcaga gaagggagcg gacatatgtc ctccgagacc ggccccgtgg ccgtggaccc
     3601 gaccctgcgt cgccgcatcg agccgcacga gttcgaggtc ttcttcgacc cgcgcgagct
     3661 gcgcaaggag acctgcctgc tgtacgagat caactggggc ggccggcact ccatctggcg
     3721 ccacacctcg cagaacacca acaagcacgt cgaggtcaac ttcatcgaga agttcaccac
     3781 cgagcggtac ttctgcccga acacccgctg ctccatcacc tggttcctgt cctggtcccc
     3841 ctgcggcgag tgctcccggg ccatcaccga gttcctgtcc cgctacccgc acgtcaccct
     3901 gttcatctac atcgcccggc tgtaccacca cgccgacccg cgcaaccggc agggcctgcg
     3961 cgacctgatc tcctccggcg tcaccatcca gatcatgacc gagcaggagt ccggctactg
     4021 ctggcggaac ttcgtcaact actccccgtc caacgaggcc cactggcccc gctacccgca
     4081 cctgtgggtc cgcctgtacg tcctggagct gtactgcatc atcctgggcc tgccgccctg
     4141 cctgaacatc ctgcgtcgca agcagccgca gctgaccttc ttcaccatcg ccctccagtc
     4201 ctgccactac cagcgcctgc cgccccacat cctgtgggcc accggcctga agtccggctc
     4261 cgagaccccg ggcacctcgg agtccgccac cccggagtcc gacaagaagt actccatcgg
     4321 cctcgcgatc ggcaccaact ccgtgggctg ggcggtcatc accgacgagt acaaggtccc
     4381 ctccaagaag ttcaaggtcc tgggcaacac cgaccggcac tcgatcaaga agaacctgat
     4441 cggcgccctg ctcttcgaca gcggcgagac cgccgaggcg acccgcctga agcggaccgc
     4501 gcgtcgccgc tacacccggc gcaagaaccg catctgctac ctgcaggaaa tcttctccaa
     4561 cgagatggcc aaggtggacg actcgttctt ccaccgcctg gaggagagct tcctggtgga
     4621 ggaggacaag aagcacgagc gccacccgat cttcggcaac atcgtggacg aggtggccta
     4681 ccacgagaag taccccacca tctaccacct ccgcaagaag ctggtggact cgaccgacaa
     4741 ggcggacctg cggctcatct acctggccct cgcgcacatg atcaagttcc gcggccactt
     4801 cctcatcgag ggcgacctga acccggacaa ctccgacgtg gacaagctct tcatccagct
     4861 ggtgcagacc tacaaccagc tgttcgagga gaaccccatc aacgccagcg gcgtggacgc
     4921 caaggcgatc ctctccgcgc gcctgagcaa gtcccggcgc ctggagaacc tcatcgccca
     4981 gctgccgggc gagaagaaga acggcctctt cggcaacctg atcgcgctgt cgctcggcct
     5041 gacccccaac ttcaagagca acttcgacct ggccgaggac gcgaagctcc agctgtccaa
     5101 ggacacctac gacgacgacc tggacaacct gctcgcccag atcggcgacc agtacgcgga
     5161 cctcttcctg gccgcgaaga acctctcgga cgccatcctg ctcagcgaca tcctgcgggt
     5221 caacaccgag atcaccaagg ccccgctgtc ggcgagcatg atcaagcggt acgacgagca
     5281 ccaccaggac ctgaccctgc tcaaggccct cgtgcgccag cagctgcccg agaagtacaa
     5341 ggaaatcttc ttcgaccagt ccaagaacgg ctacgccggc tacatcgacg gcggcgcgtc
     5401 gcaggaggag ttctacaagt tcatcaagcc gatcctggag aagatggacg gcaccgagga
     5461 gctgctcgtc aagctgaacc gcgaggacct gctccgcaag cagcggacct tcgacaacgg
     5521 ctccatcccg caccagatcc acctgggcga gctccacgcc atcctccggc gccaggagga
     5581 cttctacccc ttcctgaagg acaaccgcga gaagatcgag aagatcctga ccttccgcat
     5641 cccgtactac gtcggccccc tggcccgcgg caactcccgg ttcgcgtgga tgacccggaa
     5701 gtcggaggag accatcaccc cgtggaactt cgaggaggtc gtggacaagg gcgcgtccgc
     5761 gcagtcgttc atcgagcgca tgaccaactt cgacaagaac ctcccgaacg agaaggtcct
     5821 gcccaagcac tccctgctct acgagtactt caccgtgtac aacgagctga ccaaggtcaa
     5881 gtacgtgacc gagggcatgc ggaagccggc cttcctgtcg ggcgagcaga agaaggcgat
     5941 cgtggacctg ctcttcaaga ccaaccgcaa ggtcaccgtg aagcagctga aggaggacta
     6001 cttcaagaag atcgagtgct tcgactccgt cgagatcagc ggcgtggagg accgcttcaa
     6061 cgcctccctg ggcacctacc acgacctgct caagatcatc aaggacaagg acttcctcga
     6121 caacgaggag aacgaggaca tcctggagga catcgtcctc accctgaccc tcttcgagga
     6181 ccgcgagatg atcgaggagc ggctcaagac ctacgcccac ctgttcgacg acaaggtgat
     6241 gaagcagctg aagcgtcgcc gctacaccgg ctggggccgc ctctcccgga agctgatcaa
     6301 cggcatccgg gacaagcaga gcggcaagac catcctggac ttcctcaagt ccgacggctt
     6361 cgccaaccgc aacttcatgc agctcatcca cgacgacagc ctgaccttca aggaggacat
     6421 ccagaaggcc caggtctcgg gccagggcga cagcctccac gagcacatcg ccaacctggc
     6481 gggctccccg gcgatcaaga agggcatcct ccagaccgtc aaggtcgtgg acgagctggt
     6541 caaggtgatg ggccgccaca agcccgagaa catcgtgatc gagatggccc gggagaacca
     6601 gaccacccag aagggccaga agaactcgcg cgagcggatg aagcggatcg aggagggcat
     6661 caaggagctc ggcagccaga tcctgaagga gcacccggtc gagaacaccc agctgcagaa
     6721 cgagaagctg tacctctact acctgcagaa cggccgcgac atgtacgtgg accaggagct
     6781 cgacatcaac cggctgtccg actacgacgt ggaccacatc gtgccgcagt ccttcctgaa
     6841 ggacgactcg atcgacaaca aggtcctgac ccgctcggac aagaaccggg gcaagtccga
     6901 caacgtgccc tcggaggagg tcgtgaagaa gatgaagaac tactggcgcc agctgctcaa
     6961 cgccaagctc atcacccagc gcaagttcga caacctgacc aaggccgagc ggggcggcct
     7021 gagcgagctc gacaaggcgg gcttcatcaa gcgccagctg gtcgagaccc ggcagatcac
     7081 caagcacgtg gcccagatcc tggactcccg gatgaacacc aagtacgacg agaacgacaa
     7141 gctgatccgc gaggtcaagg tgatcaccct caagagcaag ctggtctccg acttccgcaa
     7201 ggacttccag ttctacaagg tccgggagat caacaactac caccacgccc acgacgcgta
     7261 cctgaacgcc gtcgtgggca ccgcgctgat caagaagtac ccgaagctgg agtccgagtt
     7321 cgtctacggc gactacaagg tctacgacgt gcgcaagatg atcgccaaga gcgagcagga
     7381 gatcggcaag gccaccgcga agtacttctt ctactccaac atcatgaact tcttcaagac
     7441 cgagatcacc ctggccaacg gcgagatccg caagcggccc ctgatcgaga ccaacggcga
     7501 gaccggcgag atcgtctggg acaagggccg cgacttcgcc accgtccgga aggtgctgtc
     7561 gatgccgcag gtcaacatcg tgaagaagac cgaggtgcag accggcggct tcagcaagga
     7621 gtccatcctc cccaagcgca acagcgacaa gctgatcgcc cggaagaagg actgggaccc
     7681 gaagaagtac ggcggcttcg acagccccac cgtcgcctac tccgtgctgg tcgtggcgaa
     7741 ggtcgagaag ggcaagagca agaagctgaa gtccgtgaag gagctgctcg gcatcaccat
     7801 catggagcgc tcctcgttcg agaagaaccc gatcgacttc ctggaggcca agggctacaa
     7861 ggaggtcaag aaggacctca tcatcaagct gcccaagtac agcctgttcg agctggagaa
     7921 cggccgcaag cggatgctcg cctccgcggg cgagctgcag aagggcaacg agctggccct
     7981 cccgtcgaag tacgtcaact tcctgtacct cgcgtcccac tacgagaagc tgaagggctc
     8041 gcccgaggac aacgagcaga agcagctctt cgtggagcag cacaagcact acctggacga
     8101 gatcatcgag cagatcagcg agttcagcaa gcgcgtcatc ctggccgacg cgaacctcga
     8161 caaggtgctg tccgcctaca acaagcaccg cgacaagccg atccgggagc aggcggagaa
     8221 catcatccac ctgttcaccc tcaccaacct gggcgccccc gccgcgttca agtacttcga
     8281 caccaccatc gaccgcaagc ggtacacctc caccaaggag gtcctcgacg cgaccctgat
     8341 ccaccagagc atcaccggcc tgtacgagac ccgcatcgac ctgtcccagc tcggcggcga
     8401 ctccggcggc tccaccaacc tgtccgacat catcgagaag gagaccggca agcagctggt
     8461 catccaggag tccatcctga tgctgccgga ggaagtcgag gaagtcatcg gcaacaagcc
     8521 cgagtccgac atcctggtcc acaccgccta cgacgagtcc accgacgaga acgtcatgct
     8581 gctgacctcg gacgcccccg agtacaagcc ctgggccctg gtcatccagg actccaacgg
     8641 cgagaacaag atcaagatgc tgtgatctag aggatccccg ggtaccgagc tcgaattccc
     8701 cagatctaaa gttttgtcgt ctttccagac gttagtaaat gaattttctg tatgaggttt
     8761 tgctaaacaa ctttcaacag tttcagcgga gtgagaatag aaaggaacaa ctaaaggaat
     8821 tgcgaataat aattttttca cgttgaaaat ctccaaaaaa aaaggctcca aaaggagcct
     8881 ttaattgtat cggtttatca gcttgctttc gaggtgaatt tcttaaacag cttgataccg
     8941 atagttgcgc cgacaatgac aacaaccatc gcccacgcat aaccgatata ttcggtcgct
     9001 gaggcttgca gggagtcaaa ggccgctttt gcgggatctc gtcgaaggca ctagaggcct
     9061 atatgcgggg atcgaccgcg cgggtcccgg acggggaaga gcggggagct ttgccagaga
     9121 gcgacgactt ccccttgcgt tggtgattgc cggtcagggc agccatccgc catcgtcgcg
     9181 tagggtgtca caccccagga atcgcgtcac tgaacacagc agccggtagg acgaccatga
     9241 ctgagttgga caccatcgca aatccgtccg atcccgcggt gcagcggatc atcgatgtca
     9301 ccaagccgtc gcgatccaac ataaagacaa cgttgatcga ggacgtcgag cccctcatgc
     9361 acagcatcgc ggccggggtg gagttcatcg aggtctacgg cagcgacagc agtccttttc
     9421 catctgagtt gctggatctg tgcgggcggc agaacatacc ggtccgcctc atcgactcct
     9481 cgatcgtcaa ccagttgttc aagggggagc ggaaggccaa gacattcggc atcgcccgcg
     9541 tccctcgccc ggccaggttc ggcgatatcg cgagccggcg tggggacgtc gtcgttctcg
     9601 acggggtgaa gatcgtcggg aacatcggcg cgatagtacg cacgtcgctc gcgctcggag
     9661 cgtcggggat catcctggtc gacagtgaca tcaccagcat cgcggaccgg cgtctccaaa
     9721 gggccagccg aggttacgtc ttctcccttc ccgtcgttct ctccggtcgc gaggaggcca
     9781 tcgccttcat tcgggacagc ggtatgcagc tgatgacgct caaggcggat ggcgacattt
     9841 ccgtgaagga actcggggac aatccggatc ggctggcctt gctgttcggc agcgaaaagg
     9901 gtgggccttc cgacctgttc gaggaggcgt cttccgcctc ggtttccatc cccatgatga
     9961 gccagaccga gtctctcaac gtttccgttt ccctcggaat cgcgctgcac gagaggatcg
    10021 acaggaatct cgcggccaac cgataagcgc ctctgttcct cggacgctcg gttgctcgac
    10081 ctcgattcgt cagtgatgat caacaggacc cagaaatggc acgagcccgg gattggcctc
    10141 ccgggctcgc cgcccccgaa ccggcgcagc atgccgagtg aggacgtttg ctctgtgacg
    10201 agcgtacgag agaacagcgc gtcgacacca ccgcgccccg ccggaccgaa gtgccactgc
    10261 ggcaccccga tcgtccacat ccccgggaag cgccccaaaa tctactgctc ggacgcctgc
    10321 cgaaagcgcg caaagcgtct cattgcggac cagtcacgca gcgtagcaag cacgactaca
    10381 gcgggtaatc cggacgcggg ctttagtagt aagggaaaat ctccgtccac tgcaggtcag
    10441 agcgctgtaa ctggaacggg ttctacccca aacgagggtg ccgcgacagc cgggcgggag
    10501 gacccgcgcg acgagcggtt cgcccggcgg gaccggcacc agacggtgtc gctgaacgag
    10561 gcgttcaagg gctgcgggac ccggctcact gccgggacgg ccgagctgat gtggaagccg
    10621 ggcgaggcga cgtggggcaa cacgtgccgc tgcaacaacg tccacacctg cccgtggtgc
    10681 atgagccgca tcctggccgt acggggctcg aacgtgcagc tcgccgccga cggtctcgcg
    10741 gacgccgggt acgggctgca ccttggcacg aacacgttgc gccacttcga gcgcatggcg
    10801 ttcggcaccg tccgcaaggg aatgcggcac ggcctggtcg cggtcttgca cgacggctgg
    10861 aagggcgcgt acggctcgtc cggccgccgc tggcgcacga tgcgcgacga cttcgggatc
    10921 atcggctacg agcgggcctt cgaggacacc ttcggctggg gctccggctg gcacctgcac
    10981 tggcacacgc tctgggtcac ccgcgaggtg ctcggcccgg acgcccaggc cgcgttccgc
    11041 gacgcactcg ccggagcctg ggccgccggg gtcgaatccg cgggcggcta caccgtcagc
    11101 gagacgtgcg accggcccgg ttgctcctgt gagggcaagg gccacggcac cgacgtgcgc
    11161 ccactcaacg gcgcggacgc cgctgacggc gatgcgggca agcaggcccg ctacctctac
    11221 aaggacggcg acaagaccaa gggcggcgtc gccaagatcg gcctggagct ggccggacag
    11281 aacttcaagg ccggtcgcgg cgacgaccgc atgggaccgc tcgacctggg cgacgcggcg
    11341 gccgccgagc tgcagcggct tcgtcggccc ggcccgttcg tcgagaagta ccgcgagcgc
    11401 gagttcggcg tcttccaggt ccgcaagcac taccggtcgc agaacctgaa ccggctgatc
    11461 aaggaactcg gcatccagca ggacgtccgc accgaggagg agatcaccga cgacaccgag
    11521 ggcctggtcg ccatcgcggt catcccggcc tacatctggt accgatacat cgcccgcgtc
    11581 gccggacgtc gcctcgacct catcaaggtc gccgagacgt acggcctacc gggcgtccgg
    11641 cggctcgtcg agtcctgggg cctggtgtgg ggcaaggacg tcctggaccc gcccgccccg
    11701 gaagcgccgg ccgcgccggg cgaccttgac gctgaccaga tgaggttcga ggtcatgtcc
    11761 gaggaggaag ctgctttccg cgaggcgcgc cggaaggcca acgaggcccg cacggaggag
    11821 ctggccgctt cgctcgaccg agtgcgtcag ccgaagaagg aggcgatccg cccgacgatc
    11881 agccttcgga agcgcctgaa gccgaagccg gtcaccgtcg acgtcaagac tcccccgccc
    11941 ggcgccgcgt cgcccgtgtg ccgccgctgc aagggcaagc tcgccccggt actccagccc
    12001 tggggccagc accccggaga ctgcctccgc gtcgacacag cggtcgcgta gggccgctga
    12061 gggaccgcca tgagaccgcc accccggaca tccgggtggc ggtctcatgg cggtcccggc
    12121 gcgaaagccg agggtctgcc tgccgtgagg tggccggcgg acctgggtta ctgctggaat
    12181 cccgcgccca gtccgagcgt ccgccggcgc ttttcacctg gctccgggtg ctcgtcgatg
    12241 agcacca
//