pyEvolvR (enCas9-PolI5M)

LOCUS      pyEvolvR (enCas9-PolI5M)              14085 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses enCas9 fused to PolI5M in S. cerevisiae; contains a 
            BsmbI-flanked GFP cassette for sgRNA cloning..
ACCESSION   .
VERSION     .
KEYWORDS    pyEvolvR (enCas9-PolI5M)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14085)
  AUTHORS   Tou CJ, Schaffer DV, Dueber JE
  TITLE     Targeted Diversification in the S. cerevisiae Genome with 
            CRISPR-Guided DNA Polymerase I.
  JOURNAL   ACS Synth Biol. 2020 Jun 16. doi: 10.1021/acssynbio.0c00149.
  PUBMED    32485105
REFERENCE   2  (bases 1 to 14085)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..14085
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     RBS             675..686
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             693..1406
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1418..1489
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1505..1532
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1546..1621
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1989..2396
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2397..3491
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     misc_feature    3973..4476
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(4520..5335)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5243..5262
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5327..5346
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5560..6148
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6049..6068
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6583..6990
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             7011..7031
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7059..8774
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             11181..11201
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11208..11228
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     RBS             12008..12016
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
ORIGIN
        1 agcttttgat taagccttct agtccaaaaa acacgttttt ttgtcattta tttcattttc
       61 ttagaatagt ttagtttatt cattttatag tcacgaatgt tttatgattc tatatagggt
      121 tgcaaacaag catttttcat tttatgttaa aacaatttca ggtttacctt ttattctgct
      181 tgtggtgacg cgtgtatccg cccgctcttt tggtcaccca tgtatgctgc caaaccagat
      241 gtcaacacag ctacaacgat gtgcttcagt attacatttt ttgccttcaa cgccttgatt
      301 gttctatttt tgctaataat aaatctattt catcggacta aaagtccatt agttgtaagc
      361 ggatttagct cagttgggag agcgccagac tgaagaaaaa cttcggtcaa gtcatctgga
      421 ggtcctgtgt tcgatccaca gaattcgcag atggccggca tggtcccagc ctcctcgctg
      481 gcgccggctg ggcaacacct tcgggtggcg aatgggactt gagacggaaa gtgaaacgtg
      541 atttcatgcg tcattttgaa cattttgtaa atcttattta ataatgtgtg cggcaattca
      601 catttaattt atgaatgttt tcttaacatc gcggcaactc aagaaacggc aggttcggat
      661 cttagctact agagaaagag gagaaatact agatgcgtaa aggcgaagag ctgttcactg
      721 gtgtcgtccc tattctggtg gaactggatg gtgatgtcaa cggtcataag ttttccgtgc
      781 gtggcgaggg tgaaggtgac gcaactaatg gtaaactgac gctgaagttc atctgtacta
      841 ctggtaaact gccggttcct tggccgactc tggtaacgac gctgacttat ggtgttcagt
      901 gctttgctcg ttatccggac catatgaagc agcatgactt cttcaagtcc gccatgccgg
      961 aaggctatgt gcaggaacgc acgatttcct ttaaggatga cggcacgtac aaaacgcgtg
     1021 cggaagtgaa atttgaaggc gataccctgg taaaccgcat tgagctgaaa ggcattgact
     1081 ttaaagagga cggcaatatc ctgggccata agctggaata caattttaac agccacaatg
     1141 tttacatcac cgccgataaa caaaaaaatg gcattaaagc gaattttaaa attcgccaca
     1201 acgtggagga tggcagcgtg cagctggctg atcactacca gcaaaacact ccaatcggtg
     1261 atggtcctgt tctgctgcca gacaatcact atctgagcac gcaaagcgtt ctgtctaaag
     1321 atccgaacga gaaacgcgat catatggttc tgctggagtt cgtaaccgca gcgggcatca
     1381 cgcatggtat ggatgaactg tacaaatgac caggcatcaa ataaaacgaa aggctcagtc
     1441 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca
     1501 cactggctca ccttcgggtg ggcctttctg cgtttatacg tctcagtttt agagctagaa
     1561 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     1621 ctttttttat tttttgtcac tattgttatg taaaatgcca cctctgacag tatggaacgc
     1681 aaacttctgt ctagtggata gctgagcagt tacagagatg ttacgaacca ctagtgcact
     1741 gcagtacatc gaggagaact tctagtatat ctacatacct aatattattg ccttattaaa
     1801 aatggaatcc caacaattac atcaaaatcc acattctctt caaaatcaat tgtcctgtac
     1861 ttccttgttc atgtgtgttc aaaaacgtta tatttatagg ataattatac tctatttctc
     1921 aacaagtaat tggttgtttg gccgagcggt ctaaggcgcc tgattcaaga aatatcttga
     1981 ccgcagttaa ctgtgggaat actcaggtat cgtaagatgc aagagttcga atctcttagc
     2041 aaccattatt tttttcctca acataacgag aacacacagg ggcgctatcg cacagaatca
     2101 aattcgatga ctggaaattt tttgttaatt tcagaggtcg cctgacgcat ataccttttt
     2161 caactgaaaa attgggagaa aaaggaaagg tgagagcgcc ggaaccggct tttcatatag
     2221 aatagagaag cgttcatgac taaatgcttg catcacaata cttgaagttg acaatattat
     2281 ttaaggacct attgtttttt ccaataggtg gttagcaatc gtcttacttt ctaacttttc
     2341 ttacctttta catttcagca atatatatat atatatttca aggatatacc attctaatgt
     2401 ctgcccctaa gaagatcgtc gttttgccag gtgaccacgt tggtcaagaa atcacagccg
     2461 aagccattaa ggttcttaaa gctatttctg atgttcgttc caatgtcaag ttcgatttcg
     2521 aaaatcattt aattggtggt gctgctatcg atgctacagg tgttccactt ccagatgagg
     2581 cgctggaagc ctccaagaag gctgatgccg ttttgttagg tgctgtgggt ggtcctaaat
     2641 ggggaaccgg tagtgttaga cctgaacaag gtttactaaa aatccgtaaa gaacttcaat
     2701 tgtacgccaa cttaagacca tgtaactttg catccgactc tcttttagac ttatctccaa
     2761 tcaagccaca atttgctaaa ggtactgact tcgttgttgt cagagaatta gtgggaggta
     2821 tttactttgg taagagaaag gaagacgatg gtgatggtgt cgcttgggat agtgaacaat
     2881 acaccgttcc agaagtgcaa agaatcacaa gaatggccgc tttcatggcc ctacaacatg
     2941 agccaccatt gcctatttgg tccttggata aagctaatgt tttggcctct tcaagattat
     3001 ggagaaaaac tgtggaggaa accatcaaga acgaatttcc tacattgaag gttcaacatc
     3061 aattgattga ttctgccgcc atgatcctag ttaagaaccc aacccaccta aatggtatta
     3121 taatcaccag caacatgttt ggtgatatca tctccgatga agcctccgtt atcccaggtt
     3181 ccttgggttt gttgccatct gcgtccttgg cctctttgcc agacaagaac accgcatttg
     3241 gtttgtacga accatgccac ggttctgctc cagatttgcc aaagaataag gtcaacccta
     3301 tcgccactat cttgtctgct gcaatgatgt tgaaattgtc attgaacttg cctgaagaag
     3361 gtaaggccat tgaagatgca gttaaaaagg ttttggatgc aggtatcaga actggtgatt
     3421 taggtggttc caacagtacc accgaagtcg gtgatgctgt cgccgaagaa gttaagaaaa
     3481 tccttgctta aaaagattct ctttttttat gatatttgta cataaacttt ataaatgaaa
     3541 ttcataatag aaacgacacg aaattacaaa atggaatatg ttcatagggt agacgaaact
     3601 atatacgcaa tctacataca tttatcaaga aggagaaaaa ggaggatgta aaggaataca
     3661 ggtaagcaaa ttgatactaa tggctcaacg tgataaggaa aaagaattgc actttaacat
     3721 taatattgac aaggaggagg gcaccacaca aaaagttagg tgtaacagaa aatcatgaaa
     3781 ctatgattcc taatttatat attggaggat tttctctaaa aaaaaaaaaa tacaacaaat
     3841 aaaaaacact caatgacctg accatttgat ggagtttaag tcaatacctt cttgaaccat
     3901 ttcccataat ggtgaaagtt ccctcaagaa ttttactctg tcagaaacgg ccttaacgac
     3961 gtagtcgaga gtatcacgtg ctataaaaat aattataatt taaatttttt aatataaata
     4021 tataaattaa aaatagaaag taaaaaaaga aattaaagaa aaaatagttt ttgttttccg
     4081 aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct tgctcttcct
     4141 gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaaaacc acacacgaaa
     4201 atcctgtgat tttacatttt acttatcgtt aatcgaatgt atatctattt aatctgcttt
     4261 tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaatttttt aaacctttgt
     4321 ttattttttt ttcttcattc cgtaactctt ctaccttctt tatttacttt ctaaaatcca
     4381 aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc catcattaaa
     4441 agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc ccgagcggcc gcgtgttaca
     4501 accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat
     4561 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     4621 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     4681 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     4741 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc
     4801 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     4861 cgttattcat tcgtgattgc gcctgagcga ggcgaaatac gcgatcgctg ttaaaaggac
     4921 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     4981 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     5041 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     5101 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     5161 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     5221 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     5281 tgttggaatt taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac
     5341 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat
     5401 cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa
     5461 cttttgctga gttgaaggat cagtcatgac caaaatccct taacgtgagt tttcgttcca
     5521 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5581 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5641 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5701 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5761 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5821 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5881 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5941 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6001 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6061 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6121 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6181 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6241 taaccgtgcg gccgcccctg aattcgcatc tagactgaac tggccgataa ttgcagacga
     6301 acgccttgcc aacagggagt tcttcagaga catggaggct caaaacgaaa ttattgacag
     6361 cctagacatc aatagtcata caacagaaag cgaccaccca actttggctg ataatagcgt
     6421 ataaacaatg catactttgt acgttcaaaa tacaatgcag tagatatatt tatgcatatt
     6481 acatataata catatcacat aggaagcaac aggcgcgttg gacttttaat tttcgaggac
     6541 cgcgaatcct tacatcacac ccaatccccc acaagtgatc ccccacacac catagcttca
     6601 aaatgtttct actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac
     6661 cacttcaaaa cacccaagca cagcatacta aatttcccct ctttcttcct ctagggtgtc
     6721 gttaattacc cgtactaaag gtttggaaaa gaaaaaagac accgcctcgt ttctttttct
     6781 tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaaa tttttttttt
     6841 tgattttttt ctctttcgat gacctcccat tgatatttaa gttaataaac ggtcatcaat
     6901 ttctcaagtt tcagtttcat ttttcttgtt ctattacaac tttttttact tcttgctcat
     6961 tagaaagaaa gcatagcaat ctaatctaag ttttaattac aaatatggcc ccaaagaaga
     7021 agcggaaggt cggtatccac ggagtcccag gatccggaga caagaagtac agcatcggcc
     7081 tggccatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca
     7141 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg
     7201 gcgccctgct gttcgacagc ggagaaacag ccgaggccac ccggctgaag agaaccgcca
     7261 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg
     7321 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag
     7381 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc
     7441 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg
     7501 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc
     7561 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg
     7621 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca
     7681 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc
     7741 tgcccggcga gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga
     7801 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg
     7861 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc
     7921 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga
     7981 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc
     8041 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag
     8101 agattttctt cgaccagagc aagaacggct acgccggcta catcgatggc ggagccagcc
     8161 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac
     8221 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca
     8281 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt
     8341 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc
     8401 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga
     8461 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gccagcgccc
     8521 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc
     8581 ccaagcacag cctgctgtac gagtacttca ccgtgtacaa cgagctgacc aaagtgaaat
     8641 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaagccatcg
     8701 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact
     8761 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg
     8821 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca
     8881 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca
     8941 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga
     9001 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg
     9061 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg
     9121 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc
     9181 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg
     9241 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga
     9301 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga
     9361 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca
     9421 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg
     9481 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg
     9541 acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctggcag
     9601 acgactccat cgataacaaa gtgctgactc ggagcgacaa gaaccggggc aagagcgaca
     9661 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcgccag ctgctgaatg
     9721 ccaagctgat tacccagagg aagttcgaca atctgaccaa ggccgagaga ggcggcctga
     9781 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa
     9841 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aacgacaaac
     9901 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg
     9961 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc
    10021 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc tgcgctggaa agcgagttcg
    10081 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa
    10141 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg
    10201 agattaccct ggccaacggc gagatccgga aggcgcctct gatcgagaca aacggcgaaa
    10261 caggcgagat cgtgtgggat aagggccggg actttgccac cgtgcggaaa gtgctgtcta
    10321 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt
    10381 ctatcctgcc caagaggaac agcgacaagc tgatcgccag aaagaaggac tgggacccta
    10441 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag
    10501 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca
    10561 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag
    10621 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg
    10681 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc
    10741 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc
    10801 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caaacactac ctggacgaga
    10861 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca
    10921 aggtgctgag cgcctacaac aagcacagag acaagcctat cagagagcag gccgagaata
    10981 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca
    11041 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc
    11101 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgacg
    11161 ccagcctggg cagcggctcc cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc
    11221 ggaaagtgga cagtgaaacc ccgggaacaa gtgagtcggc cacccctgaa ggtggatcag
    11281 ggggtagcgg atccgttcag atcccgcaga acccgctgat tctggttgac ggatctagtt
    11341 acctgtaccg tgcttaccat gctttcccgc ctttgaccaa ttctgctggt gaacctacgg
    11401 gagctatgta cggagttctg aatatgttgc gttctttaat tatgcagtac aagcctaccc
    11461 acgctgctgt tgttttcgat gctaaaggta agacgttccg cgacgagtta ttcgagcact
    11521 ataagtctca ccgtcctccg atgcctgatg acttacgcgc tcagattgag ccgctgcatg
    11581 ctatggtgaa ggctatgggt ttacctcttt tggctgtcag cggtgttgag gctgatgatg
    11641 tcattggcac cttagctcgt gaggctgaga aggctggtcg ccctgttttg atttctaccg
    11701 gtgacaagga catggctcaa ttggttaccc cgaacatcac cctgatcaac accatgacca
    11761 acacgattct gggtcctgag gaagttgtta acaaatatgg tgttcctccg gagttgatta
    11821 ttgactttct tgctcttatg ggcgattctt cagacaatat cccgggtgtt ccaggtgttg
    11881 gagagaagac tgctcaagct ctgcttcagg gtctgggtgg tttggacacc ctttacgctg
    11941 aaccggagaa gatcgccggt ctgtcttttc gcggtgctaa gaccatggct gctaaactgg
    12001 aacagaataa ggaggtcgca tacctgtctt atcaattggc taccatcaag acggatgtgg
    12061 agttagaact tacgtgcgag cagcttgagg ttcaacagcc tgctgctgag gaactgctgg
    12121 gtctttttaa gaaatacgaa tttaagcgtt ggaccgccga cgttgaggct ggtaagtggc
    12181 tgcaagctaa gggtgctaag ccggctgcta aaccgcaaga aacgagtgtc gctgatgagg
    12241 ctccggaggt taccgctacc gttatctctt acgataatta tgttacgatt ctggacgagg
    12301 aaaccttaaa ggcttggatc gctaaattag agaaggctcc tgttttcgct ttcgacacgg
    12361 aaacggattc tctggacaat attagtgcga atcttgttgg tctgagtttc gcaattgaac
    12421 cgggtgttgc tgcttacatc cctgtggcac acgactacct ggacgctccg gaccagattt
    12481 cacgtgaacg cgctctggaa ctgctgaagc ctttattaga ggacgagaaa gctttgaaag
    12541 ttggtcagaa tttgaagtat gctcgtggaa tcttagctaa ttatggtatc gagttgcgcg
    12601 gtatcgcttt cgacacgatg ttggaatctt atatcctgaa ctctgtcgct ggtcgccatg
    12661 acatggactc tctggctgag cgctggctga aacataagac gattaccttc gaggaaatcg
    12721 caggaaaggg taagaaccag ctcacgttca atcaaatcgc tctggaggaa gctggtcgct
    12781 atgctgctga ggacgctgac gttactctgc aactgcactt gaagatgtgg cctgacttgc
    12841 agaagcataa gggtccactg aatgtttttg aaaacattga gatgcctttg gttccagttc
    12901 tgtctcgtat cgagcgcaat ggcgttaaaa ttgacccaaa ggttttacat aaccactcag
    12961 aggaactgac gctgcgctta gccgaattgg agaaaaaggc tcacgagatc gctggcgaag
    13021 agttcaatct gtcatctacg aaacaactgc agactatcct gttcgagaag caaggtatca
    13081 agccattaaa aaagacccct ggcggtgctc cgtctacctc tgaggaagtt ttggaggagt
    13141 tagctttgga ttaccctctg ccgaaggtta tcttggaata ccgcggtttg gctaaattga
    13201 agtctactta tacggataaa cttcctttga tgattaatcc aaagacgggt cgcgttcaca
    13261 cgtcgtacca tcaagctgtt accgctaccg gtcgcctgtc ttctacggat ccgaatttac
    13321 agaatattcc tgtgcgcaat gaggagggcc gccgcattcg tcaagctttt atcgctccgg
    13381 aagactacgt tatcgtttct gctgattatt ctcaaaatga attacgtatc atggctcacc
    13441 tgtctcgcga taagggtctg ttgacggcct ttgctgaggg taaggacatt catcgtgcta
    13501 ccgctgctga ggtttacggc ctgccgttgg aaacggttac gtctgaacag cgtcgctctg
    13561 ctaagcgtat taatttcggc ttaatctacg gtatgtctgc gtttggctta gctcgtcagc
    13621 tgaatatccc gcgcaaggaa gctcaaaaat atatggatct gtattttgag cgttaccacg
    13681 gtgttttgga atacatggag cgtacgcgcg cgcaagctaa ggaacaaggt tatgtggaaa
    13741 ccttggatgg tcgtcgcttg tacttgcctg acattaagtc ttctaacggc gcccgccgcg
    13801 ctgctgccga gcgcgctgct atcaatgctc cgatgcaagg tactgctgct gatattatta
    13861 agcgtgctat gatcgctgtg gacgcttggc tgcaagctga acagcctcgc gttcgcatga
    13921 ttatgcaagt tcatgacgag ttggttttcg aggtgcataa ggacgacgtg gacgctgttg
    13981 ctaaacaaat ccaccagttg atggagaatt gcacgcgctt agacgttccg ctgctggttg
    14041 aagttggttc tggtgaaaac tgggaccagg ctcactaata atggc
//