pCBEclos-cbei2385-g2-opt

LOCUS       pCBEclos-cbei2385-g2-opt             10690 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPR-mediated base editing in Clostridium.
ACCESSION   .
VERSION     .
KEYWORDS    pCBEclos-cbei2385-g2-opt
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10690)
  AUTHORS   Li Q, Seys FM, Minton NP, Yang J, Jiang Y, Jiang W, Yang S
  TITLE     CRISPR-Cas9(D10A) nickase-assisted base editing in the solvent 
            producer Clostridium beijerinckii.
  JOURNAL   Biotechnol Bioeng. 2019 Feb 10. doi: 10.1002/bit.26949.
  PUBMED    30739328
REFERENCE   2  (bases 1 to 10690)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..10690
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1486..1520
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1541..1616
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1788..2474
                     /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             2523..6623
                     /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             6636..6884
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     primer_bind     complement(6911..6928)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6911..6927)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6920..6942)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7136..7155)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7255..7277
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7315..7333)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7401..7505
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7506..8366
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7724..7743)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8537..9125
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9026..9045
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9279..9296
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9413..9434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        9449..9479
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9487..9503
                     /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     9492..9514
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9511..9527
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9511..9527
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cgataattta cagaaaagaa aattatagaa tttagtatga ttaattatac tcatttatga
       61 atgtttaatt gaatacaaaa aaaaatactt gttatgtatt caattacggg ttaaaatata
      121 gacaagttga aaaatttaat aaaaaaataa gtcctcagct cttatatatt aagctaccaa
      181 cttagtatat aagccaaaac ttaaatgtgc taccaacaca tcaagccgtt agagaactct
      241 atctatagca atatttcaaa tgtaccgaca tacaagagaa acattaacta tatatattca
      301 atttatgaga ttatcttaac agatataaat gtaaattgca ataagtaaga tttagaagtt
      361 tatagccttt gtgtattgga agcagtacgc aaaggctttt ttatttgata aaaattagaa
      421 gtatatttat tttttcataa ttaatttatg aaaatgaaag ggggtgagca aagtgacaga
      481 ggaaagcagt atcttatcaa ataacaaggt attagcaata tcattattga ctttagcagt
      541 aaacattatg acttttatag tgcttgtagc taagtagtac gaaaggggga gctttaaaaa
      601 gctccttgga atacatagaa ttcataaatt aatttatgaa aagaagggcg tatatgaaaa
      661 cttgtaaaaa ttgcaaagag tttattaaag atactgaaat atgcaaaata cattcgttga
      721 tgattcatga taaaacagta gcaacctatt gcagtaaata caatgagtca agatgtttac
      781 ataaagggaa agtccaatgt attaattgtt caaagatgaa ccgatatgga tggtgtgcca
      841 taaaaatgag atgttttaca gaggaagaac agaaaaaaga acgtacatgc attaaatatt
      901 atgcaaggag ctttaaaaaa gctcatgtaa agaagagtaa aaagaaaaaa taatttattt
      961 attaatttaa tattgagagt gccgacacag tatgcactaa aaaatatatc tgtggtgtag
     1021 tgagccgata caaaaggata gtcactcgca ttttcataat acatcttatg ttatgattat
     1081 gtgtcggtgg gacttcacga cgaaaaccca caataaaaaa agagttcggg gtagggttaa
     1141 gcatagttga ggcaactaaa caatcaagct aggatatgca gtagcagacc gtaaggtcgt
     1201 tgtttaggtg tgttgtaata catacgctat taagatgtaa aaatacggat accaatgaag
     1261 ggaaaagtat aatttttgga tgtagtttgt ttgttcatct atgggcaaac tacgtccaaa
     1321 gccgtttcca aatctgctaa aaagtatatc ctttctaaaa tcaaagtcaa gtatgaaatc
     1381 ataaataaag tttaattttg aagttattat gatattatgt ttttctatta aaataaatta
     1441 agtatataga atagtttaat aatagtatat acttaatgtg ctgcattgac agctagctca
     1501 gtcctaggta taatactagt cttattccca tttcttcttt gttttagagc tagaaatagc
     1561 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     1621 ttggtacctt tttaacaaaa tatattgata aaaataataa tagtgggtat aattaagttg
     1681 ttagagaaaa cgtataaatt agggataaac tatggaactt atgaaataga ttgaaatggt
     1741 ttatctgtta ccccgtatca aaatttagga ggttagttag aggatccatg tcaagtgaaa
     1801 caggaccagt tgctgtagat cctactctta gaagaagaat agaaccacat gaatttgaag
     1861 ttttctttga tcctagagaa cttagaaaag aaacatgtct tctttatgaa ataaattggg
     1921 gaggaagaca ttcaatatgg agacatacaa gtcaaaatac taataaacat gttgaagtaa
     1981 attttataga aaaatttaca actgaaagat atttttgtcc aaatacaaga tgttcaataa
     2041 cttggttttt aagttggtct ccttgtggag aatgttctag agctataaca gaatttttat
     2101 caagatatcc acatgtaact ctttttatat atatagcaag actttatcat catgctgatc
     2161 ctagaaatag acaaggactt agagatctta tatcttcagg agttacaata caaataatga
     2221 ctgaacaaga aagtggatat tgttggagaa attttgtaaa ttattcacca agtaatgaag
     2281 cacattggcc aagatatcct catctttggg ttagacttta tgtacttgaa ctttattgta
     2341 taatattagg attaccacct tgtttaaata tattaagaag aaaacaacct caacttacat
     2401 ttttcactat agcacttcaa agttgtcatt atcaaagatt accacctcat atactttggg
     2461 ctacaggatt aaaatctgga tcagaaacac caggaacatc agaatctgca actcctgaat
     2521 ctgataagaa atactcaata ggcttagcta tcggcacaaa tagcgtcgga tgggcggtga
     2581 tcactgatga atataaggtt ccgtctaaaa agttcaaggt tctgggaaat acagaccgcc
     2641 acagtatcaa aaaaaatctt ataggggctc ttttatttga cagtggagag acagcggaag
     2701 cgactcgtct caaacggaca gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt
     2761 atctacagga gattttttca aatgagatgg cgaaagtaga tgatagtttc tttcatcgac
     2821 ttgaagagtc ttttttggtg gaagaagaca agaagcatga acgtcatcct atttttggaa
     2881 atatagtaga tgaagttgct tatcatgaga aatatccaac tatctatcat ctgcgaaaaa
     2941 aattggtaga ttctactgat aaagcggatt tgcgcttaat ctatttggcc ttagcgcata
     3001 tgattaagtt tcgtggtcat tttttgattg agggagattt aaatcctgat aatagtgatg
     3061 tggacaaact atttatccag ttggtacaaa cctacaatca attatttgaa gaaaacccta
     3121 ttaacgcaag tggagtagat gctaaagcga ttctttctgc acgattgagt aaatcaagac
     3181 gattagaaaa tctcattgct cagctccccg gtgagaagaa aaatggctta tttgggaatc
     3241 tcattgcttt gtcattgggt ttgaccccta attttaaatc aaattttgat ttggcagaag
     3301 atgctaaatt acagctttca aaagatactt acgatgatga tttagataat ttattggcgc
     3361 aaattggaga tcaatatgct gatttgtttt tggcagctaa gaatttatca gatgctattt
     3421 tactttcaga tatcctaaga gtaaatactg aaataactaa ggctccccta tcagcttcaa
     3481 tgattaaacg ctacgatgaa catcatcaag acttgactct tttaaaagct ttagttcgac
     3541 aacaacttcc agaaaagtat aaagaaatct tttttgatca atcaaaaaac ggatatgcag
     3601 gttatattga tgggggagct agccaagaag aattttataa atttatcaaa ccaattttag
     3661 aaaaaatgga tggtactgag gaattattgg tgaaactaaa tcgtgaagat ttgctgcgca
     3721 agcaacggac ctttgacaac ggctctattc cccatcaaat tcacttgggt gagctgcatg
     3781 ctattttgag aagacaagaa gacttttatc catttttaaa agacaatcgt gagaagattg
     3841 aaaaaatctt gacttttcga attccttatt atgttggtcc attggcgcgt ggcaatagtc
     3901 gttttgcatg gatgactcgg aagtctgaag aaacaattac cccatggaat tttgaagaag
     3961 ttgtcgataa aggtgcttca gctcaatcat ttattgaacg catgacaaac tttgataaaa
     4021 atcttccaaa tgaaaaagta ctaccaaaac atagtttgct ttatgagtat tttacggttt
     4081 ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat gcgaaaacca gcatttcttt
     4141 caggtgaaca gaagaaagcc attgttgatt tactcttcaa aacaaatcga aaagtaaccg
     4201 ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg ttttgatagt gttgaaattt
     4261 caggagttga agatagattt aatgcttcat taggtaccta ccatgatttg ctaaaaatta
     4321 ttaaagataa agattttttg gataatgaag aaaatgaaga tatcttagag gatattgttt
     4381 taacattgac cttatttgaa gatagggaga tgattgagga aagacttaaa acatatgctc
     4441 acctctttga tgataaggtg atgaaacagc ttaaacgtcg ccgttatact ggttggggac
     4501 gtttgtctcg aaaattgatt aatggtatta gggataagca atctggcaaa acaatattag
     4561 attttttgaa atcagatggt tttgccaatc gcaattttat gcagctgatc catgatgata
     4621 gtttgacatt taaagaagac attcaaaaag cacaagtgtc tggacaaggc gatagtttac
     4681 atgaacatat tgcaaattta gctggtagcc ctgctattaa aaaaggtatt ttacagactg
     4741 taaaagttgt tgatgaattg gtcaaagtaa tggggcggca taagccagaa aatatcgtta
     4801 ttgaaatggc acgtgaaaat cagacaactc aaaagggcca gaaaaattcg cgagagcgta
     4861 tgaaacgaat cgaagaaggt atcaaagaat taggaagtca gattcttaaa gagcatcctg
     4921 ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta ttatctccaa aatggaagag
     4981 acatgtatgt ggaccaagaa ttagatatta atcgtttaag tgattatgat gtcgatcaca
     5041 ttgttccaca aagtttcctt aaagacgatt caatagacaa taaggtctta acgcgttctg
     5101 ataaaaatcg tggtaaatcg gataacgttc caagtgaaga agtagtcaaa aagatgaaaa
     5161 actattggag acaacttcta aacgccaagt taatcactca acgtaagttt gataatttaa
     5221 cgaaagctga acgtggaggt ttgagtgaac ttgataaagc tggttttatc aaacgccaat
     5281 tggttgaaac tcgccaaatc actaagcatg tggcacaaat tttggatagt cgcatgaata
     5341 ctaaatacga tgaaaatgat aaacttattc gagaggttaa agtgattacc ttaaaatcta
     5401 aattagtttc tgacttccga aaagatttcc aattctataa agtacgtgag attaacaatt
     5461 accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg aactgctttg attaagaaat
     5521 atccaaaact tgaatcggag tttgtctatg gtgattataa agtttatgat gttcgtaaaa
     5581 tgattgctaa gtctgagcaa gaaataggca aagcaaccgc aaaatatttc ttttactcta
     5641 atatcatgaa cttcttcaaa acagaaatta cacttgcaaa tggagagatt cgcaaacgcc
     5701 ctctaatcga aactaatggg gaaactggag aaattgtctg ggataaaggg cgagattttg
     5761 ccacagtgcg caaagtattg tccatgcccc aagtcaatat tgtcaagaaa acagaagtac
     5821 agacaggcgg attctccaag gagtcaattt taccaaaaag aaattcggac aagcttattg
     5881 ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt tgatagtcca acggtagctt
     5941 attcagtcct agtggttgct aaggtggaaa aagggaaatc gaagaagtta aaatccgtta
     6001 aagagttact agggatcaca attatggaaa gaagttcctt tgaaaaaaat ccgattgact
     6061 ttttagaagc taaaggatat aaggaagtta aaaaagactt aatcattaaa ctacctaaat
     6121 atagtctttt tgagttagaa aacggtcgta aacggatgct ggctagtgcc ggagaattac
     6181 aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa ttttttatat ttagctagtc
     6241 attatgaaaa gttgaagggt agtccagaag ataacgaaca aaaacaattg tttgtggagc
     6301 agcataagca ttatttagat gagattattg agcaaatcag tgaattttct aagcgtgtta
     6361 ttttagcaga tgccaattta gataaagttc ttagtgcata taacaaacat agagacaaac
     6421 caatacgtga acaagcagaa aatattattc atttatttac gttgacgaat cttggagctc
     6481 ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa acgatatacg tctacaaaag
     6541 aagttttaga tgccactctt atccatcaat ccatcactgg tctttatgaa acacgcattg
     6601 atttgagtca gctaggaggt gacagtggag gatcaacaaa tctttcagat ataatagaaa
     6661 aagaaactgg aaaacaatta gttatacaag aaagtatact tatgcttcca gaagaagttg
     6721 aagaagtaat aggaaataaa cctgaatctg atatattagt tcatacagca tatgatgaaa
     6781 gtactgatga aaatgtaatg cttcttacat ctgatgcacc agaatataaa ccttgggctt
     6841 tagtaataca agattcaaat ggagaaaata aaataaaaat gttatctgga ggatcataac
     6901 ccgggaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     6961 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     7021 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt
     7081 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     7141 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     7201 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     7261 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     7321 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     7381 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     7441 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     7501 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     7561 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     7621 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     7681 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     7741 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     7801 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     7861 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     7921 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     7981 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     8041 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     8101 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     8161 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     8221 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     8281 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     8341 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     8401 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     8461 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     8521 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     8581 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     8641 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     8701 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     8761 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     8821 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     8881 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     8941 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     9001 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     9061 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     9121 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     9181 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     9241 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     9301 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     9361 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     9421 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     9481 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     9541 agctttggct aacacacacg ccattccaac caatagtttt ctcggcataa agccatgctc
     9601 tgacgcttaa atgcactaat gccttaaaaa aacattaaag tctaacacac tagacttatt
     9661 tacttcgtaa ttaagtcgtt aaaccgtgtg ctctacgacc aaaagtataa aacctttaag
     9721 aactttcttt tttcttgtaa aaaaagaaac tagataaatc tctcatatct tttattcaat
     9781 aatcgcatca gattgcagta taaatttaac gatcactcat catgttcata tttatcagag
     9841 ctcgtgctat aattatacta attttataag gaggaaaaaa taaagagggt tataatgaac
     9901 gagaaaaata taaaacacag tcaaaacttt attacttcaa aacataatat agataaaata
     9961 atgacaaata taagattaaa tgaacatgat aatatctttg aaatcggctc aggaaaaggg
    10021 cattttaccc ttgaattagt acagaggtgt aatttcgtaa ctgccattga aatagaccat
    10081 aaattatgca aaactacaga aaataaactt gttgatcacg ataatttcca agttttaaac
    10141 aaggatatat tgcagtttaa atttcctaaa aaccaatcct ataaaatatt tggtaatata
    10201 ccttataaca taagtacgga tataatacgc aaaattgttt ttgatagtat agctgatgag
    10261 atttatttaa tcgtggaata cgggtttgct aaaagattat taaatacaaa acgctcattg
    10321 gcattatttt taatggcaga agttgatatt tctatattaa gtatggttcc aagagaatat
    10381 tttcatccta aacctaaagt gaatagctca cttatcagat taaatagaaa aaaatcaaga
    10441 atatcacaca aagataaaca gaagtataat tatttcgtta tgaaatgggt taacaaagaa
    10501 tacaagaaaa tatttacaaa aaatcaattt aacaattcct taaaacatgc aggaattgac
    10561 gatttaaaca atattagctt tgaacaattc ttatctcttt tcaatagcta taaattattt
    10621 aataagtaag ttaagggatg cataaactgc atcccttaac ttgtttttcg tgtacctatt
    10681 ttttgtgaat
//