LOCUS       sequence-248750-       10232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses IgnaviCas9 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10232)
  AUTHORS   Schmidt ST, Yu FB, Blainey PC, May AP, Quake SR
  TITLE     Nucleic acid cleavage with a hyperthermophilic Cas9 from an 
            uncultured Ignavibacterium.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Oct 28. pii: 1904273116. doi: 
            10.1073/pnas.1904273116.
  PUBMED    31659048
REFERENCE   2  (bases 1 to 10232)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A. 2019 Oct 28. pii: 1904273116. doi: 
            10.1073/pnas.1904273116."
COMMENT     Sequence Label: pIgnaviCas9
FEATURES             Location/Qualifiers
     source          1..10232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..80
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    80..104
                     /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)."
     RBS             119..141
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             152..169
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             173..1270
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1244..1267
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1301..1321
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5060..5080
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5157..5174
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5227..5245)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5241..5288
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5325..5780
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5412..5431)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5622..5643
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5872..6687)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5872..6687)
                     /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     6595..6614
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6679..6698
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6809..7397
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7298..7317
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7551..7568
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7583..7722
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7648..7667)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7808..7830
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7824..8015)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(8824..9906)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     9868..9887
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9907..9984)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     10190..10209
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
       61 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
      121 tgtttaactt taagaaggag atatacatat gcaccatcac catcaccatg gaaaaatcga
      181 agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt
      241 cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact
      301 ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc
      361 acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa
      421 agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct
      481 gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc
      541 gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg
      601 taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc
      661 tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt
      721 ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca
      781 catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc
      841 gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg
      901 tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag
      961 cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta
     1021 tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc
     1081 gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca ctatggaaaa
     1141 cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt
     1201 gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga
     1261 cgcgcagact aattcgagct cgaacaacaa caacactagt gaaaacctgt atttccaggg
     1321 agcagcctcg atgaaaaaag tattaggatt agatcttgga gtatcttcaa taggctgggc
     1381 tttaattgac gaagatgata gaaaaataat gggcatgggt agtagaataa taccattaac
     1441 aactgatgat aaagacgagt ttacaaaagg caatacgatt tctaagaatc agcaacgaac
     1501 aattaaaaga actcaaagaa aaggatacga tcgttatcaa ttaagaaggc agaatttagt
     1561 tttcgtgttg aaacaaaata atatgatgcc tgatattgaa ttagtaaatc ttccaaaact
     1621 tgaattatgg aaactaagaa gtgatgcggt taataaaaaa atatctttga aagaattagg
     1681 cagaatccta cttcacttaa atcaaaaaag aggttataaa agtagcagaa gtgaatcaaa
     1741 tttggataag aaagataccg aatatgtagc aacagtaaaa aacagatatg aaagcctaaa
     1801 agaaattggt ttaacaatag gacagaaatt ttttgaggaa ttatccaaaa acaattttta
     1861 cagaataaaa gaacaggttt acccaagaga agcatatgtt gaagagtata ataaaataat
     1921 gaagcatcaa caaaaacatt atccagaaaa tatttcggaa gaattaatta ataaaataag
     1981 agacgaaata atttactatc aacgaaaact aaaatcgcaa aagggattgg tgtctgtttg
     2041 cgagtttgaa ggattttgga taaagctaaa ttcaaatgga aaagaaaaag atttatttgt
     2101 tggtccaaaa gtaactccta aaagttcacc attattccag gtaagtagaa tttgggaaac
     2161 tatcaataac atatcaatta aaagaaagac tggtgaatcc attgaaatta cactggataa
     2221 aaagaaagaa atttttgctt atatggataa aaatgaaaaa ttaagctatc cagaattatt
     2281 aaaaatttta gggcttaaaa aagatgacgt atatggaaac aagaatttaa caaatgggtt
     2341 gctgggcaac aaaataaaaa cagaaatgat gaagtgtatt tcagatattg ataagtattc
     2401 tgatttattc cgattagaac ttgaaataaa agaattcgat gaagaggttt atttatatga
     2461 tagaacaacc ggagaaataa taaattcaaa gaaaaaaaag aatataatag cagcaataga
     2521 agaccaacca ttttacaagc tttggcatgt tgtttattca atacccgata aagaaacttg
     2581 tcaaaaaata cttatgtcaa aatttggcat acaggaagaa gacgctgcta aattagcaac
     2641 acttgatttt actaaacttg gtttttcgaa caaatcccac cgtgcaatta ggaaaatgct
     2701 tccttatcta atggaagggg ataacgatta tatggcccgt tgttatgcgg gttatcatca
     2761 cacaacaaca attacaaaac aagaaaactt ccaaagaaaa ctgttagata aattaaaaaa
     2821 cttagaaaaa aatagcctgc gccagccgat agttgaaaaa attctaaatc agatgataaa
     2881 tgttgtaaat gcaattatag acaaatatgg gaaaccggat gaaattagaa ttgaactagc
     2941 cagagaatta aaacagagta gagaagaaag aaatgaagca tatagaaaca tgaatgaacg
     3001 agaacgtgaa aataaaataa ttgaaaaaga gctttctgaa tttggacttc gtgcaacacg
     3061 aaacaatatt atcaaatgga gattatatca cgaaattagc aacgaagaaa agaaacaaaa
     3121 tgcaatttgc atttattgtg gcaaaccaat ttcctttact gctgcaatat taggtgaaga
     3181 agttgaagtt gaacacataa taccaaggtc aaggttattt gacgattctc aaagcaataa
     3241 aacactggca catagaaaat gcaatgcaga taagaaagac caaacagctt atgactttat
     3301 gcgttcaaaa tctgatactg aatttaatga ttacgttgag cgaattaata ccctttataa
     3361 aaatcatgta attggaaaaa cgaaaagaga taaactttta atgtctgaag aaaaaattcc
     3421 tatggatttt attgacagac aattaagaca aacacaatac atctctaaaa aagcattaga
     3481 gcttcttcag aatatctgtt ataatgtgtg ggcaacaagc ggaaatgtga ccgccgagtt
     3541 gcgccatata tggggatggg atgaagtgct tgaaaatctt caattaccta agtatagaga
     3601 aagtggatta atagaaatta ttgaagttgg agataaagat aataaacaaa aaaaggaaaa
     3661 gataattgga tggaccaaaa gagacgatca tagacatcat gcaattgatg ctcttaccat
     3721 cgcatgtacc aaacaaggat ttatccaacg ctttaataga ttaaatagtg ggaaagtacg
     3781 aaacgatatg cttcaggaaa ttgaaaacgc caaacagaat tacgataaaa gaaaaaatct
     3841 tttggagaac tatattcttt cttacagacc atttacaaca aaggaagttg aaagagaggc
     3901 tgagaaaata cttgtatcat tcaaagccgg caaaaaggtt gcatctacag gcaaaagaaa
     3961 aattaaaaaa gatggcaaaa aaataatcgc tcaaactggt attattattc caagaggacc
     4021 attaagtgaa gaaagtgtct atggaaaaat aaaagtaatt gagaaggaaa aaccgttaaa
     4081 atatttattt gaaaatccac acctcatatt taaaccaaat ataaaagcac ttgtagaaga
     4141 aagactttac aaaaacaata acgaccctaa aagtgctata gcttcattaa aaaaagaacc
     4201 tatttatctt gacaaagaga aaacaataaa attggaatac ggaacatgtt ataaagaaga
     4261 agttgttata aaaaaaccac tacaagcttt gaacgagaag caagtagagg atattgttga
     4321 ccctataata aaacaaaaga ttaaggatcg actggttaaa tttggtggca aagccaaaga
     4381 agcatttaag gatttagaaa acgaacctat ttggtatgat gaggaaaaaa gaattccaat
     4441 aaagaatgtt cgatggttta caggactttc agcaattgaa cctataagca aggatgagac
     4501 cggaaaagaa attggatttg tcaaacctgg caataatcat catcttgcaa tatacattga
     4561 tgaagaaggg aaaaaacaac ttagtatatg ttcattttgg catgctgtag aaagaaagaa
     4621 atatgggttg cctgttataa taaaaaatcc gtcagaggtt gttgatttta tacttgcgga
     4681 ggaaaatgaa gataaatatc cagaaagttt tctagaaaaa ttacccgctg ggaaatggac
     4741 atttaaagaa agctttcaac aaaacgagat gtttgtactt ggaataagca aagaagcatt
     4801 tgaagaagcc atttcgagaa atgattatag cttcttaagt aattacttat atcgtgttca
     4861 aaagattgca atgataggca aacaaccaaa tattgttttt agacatcatc tcgaaactca
     4921 gcttaaggat gacgcatacg ctaaaaaaag taatcgcttt tatttaatac aaagtatcgg
     4981 ggcattagaa tcattatatc caataaaaat tttaattaat tgtttgggag aaattattac
     5041 taataataaa taagggtcac ctaagaaaaa acgaaaagtt gagaccggtt aacgaggccg
     5101 gtgctttgca ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc
     5161 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     5221 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     5281 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     5341 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5401 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5461 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5521 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5581 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5641 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     5701 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5761 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5821 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     5881 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     5941 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     6001 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     6061 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     6121 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     6181 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     6241 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     6301 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     6361 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     6421 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     6481 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     6541 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     6601 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     6661 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     6721 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6781 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6841 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6901 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     6961 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     7021 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     7081 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     7141 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7201 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7261 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7321 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7381 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7441 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     7501 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     7561 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     7621 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     7681 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     7741 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     7801 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     7861 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     7921 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     7981 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     8041 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     8101 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     8161 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     8221 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     8281 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     8341 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     8401 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     8461 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     8521 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     8581 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     8641 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     8701 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     8761 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     8821 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     8881 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     8941 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     9001 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     9061 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     9121 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     9181 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     9241 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     9301 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     9361 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     9421 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     9481 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     9541 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     9601 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     9661 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     9721 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     9781 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     9841 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     9901 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     9961 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
    10021 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
    10081 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
    10141 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
    10201 cgatataggc gccagcaacc gcacctgtgg cg
//
