LOCUS       sequence-272023-        8632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  obtaining full Cas9 RNP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8632)
  AUTHORS   Qiao J, Li W, Lin S, Sun W, Ma L, Liu Y
  TITLE     Co-expression of Cas9 and single-guided RNAs in Escherichia coli 
            streamlines production of Cas9 ribonucleoproteins.
  JOURNAL   Commun Biol. 2019 May 3;2:161. doi: 10.1038/s42003-019-0402-x. 
            eCollection 2019.
  PUBMED    31069270
REFERENCE   2  (bases 1 to 8632)
  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: "Commun 
            Biol."; date: "2019-05-3"; volume: "2:161. doi"; pages: " 
            10.1038/s42003-019-0402-x. eCollection 2019"
COMMENT     Sequence Label: pCold CL7-Cas9
FEATURES             Location/Qualifiers
     source          1..8632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(75..80,81..96,97..103)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    109..125
                     /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)."
     5'UTR           147..280
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             689..712
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             713..4816
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             713..4816
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4817..4837
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4847..4864
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     3'UTR           4875..5019
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     primer_bind     complement(5060..5082)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5182..5201
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5224..5242
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        5263..5338
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5374..5445
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        5551..5655
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5656..5724,5725..6516)
                     /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(5874..5893)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6687..7275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7176..7195
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7447..8529)
                     /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     8491..8510
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8530..8607)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
       61 aaggaatggt gtggttgaca attaatcatc ggctcgtata atgtgtaatt gtgagcggat
      121 aacaatttga tgtgctagcg catatccagt gtagtaaggc aagtcccttc aagagttatc
      181 gttgataccc ctcgtagtgc acattccttt aacgcttcaa aatctgtaaa gcacgccata
      241 tcgccgaaag gcacacttaa ttattaagag gtaatacacc atgagcaaaa gcaatgaacc
      301 gggtaaagca accggtgaag gtaaaccggt taataacaaa tggctgaaca atgccggtaa
      361 agatctgggt agtccggttc cggatcgtat tgcaaataaa ctgcgtgata aagaattcga
      421 gagcttcgat gattttcgtg aaaccttttg ggaagaagtt agcaaagatc ctgaactgag
      481 caaacagttt agccgcaata acaatgatcg tatgaaagtt ggtaaagcac cgaaaacacg
      541 tacccaggat gttagcggta aacgtacctc atttgaactg aatcatcaga aaccgattga
      601 acagaatggt ggcgtttatg atatggataa cattagcgtt gttaccccga aacgcaacat
      661 tgatattgaa ggcggtggtg gtggttcttt ggaggttttg ttccagggtc caatggataa
      721 gaaatactca ataggcttag atatcggcac aaatagcgtc ggatgggcgg tgatcactga
      781 tgaatataag gttccgtcta aaaagttcaa ggttctggga aatacagacc gccacagtat
      841 caaaaaaaat cttatagggg ctcttttatt tgacagtgga gagacagcgg aagcgactcg
      901 tctcaaacgg acagctcgta gaaggtatac acgtcggaag aatcgtattt gttatctaca
      961 ggagattttt tcaaatgaga tggcgaaagt agatgatagt ttctttcatc gacttgaaga
     1021 gtcttttttg gtggaagaag acaagaagca tgaacgtcat cctatttttg gaaatatagt
     1081 agatgaagtt gcttatcatg agaaatatcc aactatctat catctgcgaa aaaaattggt
     1141 agattctact gataaagcgg atttgcgctt aatctatttg gccttagcgc atatgattaa
     1201 gtttcgtggt cattttttga ttgagggaga tttaaatcct gataatagtg atgtggacaa
     1261 actatttatc cagttggtac aaacctacaa tcaattattt gaagaaaacc ctattaacgc
     1321 aagtggagta gatgctaaag cgattctttc tgcacgattg agtaaatcaa gacgattaga
     1381 aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc ttatttggga atctcattgc
     1441 tttgtcattg ggtttgaccc ctaattttaa atcaaatttt gatttggcag aagatgctaa
     1501 attacagctt tcaaaagata cttacgatga tgatttagat aatttattgg cgcaaattgg
     1561 agatcaatat gctgatttgt ttttggcagc taagaattta tcagatgcta ttttactttc
     1621 agatatccta agagtaaata ctgaaataac taaggctccc ctatcagctt caatgattaa
     1681 acgctacgat gaacatcatc aagacttgac tcttttaaaa gctttagttc gacaacaact
     1741 tccagaaaag tataaagaaa tcttttttga tcaatcaaaa aacggatatg caggttatat
     1801 tgatggggga gctagccaag aagaatttta taaatttatc aaaccaattt tagaaaaaat
     1861 ggatggtact gaggaattat tggtgaaact aaatcgtgaa gatttgctgc gcaagcaacg
     1921 gacctttgac aacggctcta ttccccatca aattcacttg ggtgagctgc atgctatttt
     1981 gagaagacaa gaagactttt atccattttt aaaagacaat cgtgagaaga ttgaaaaaat
     2041 cttgactttt cgcattcctt attatgttgg tccattggcg cgtggcaata gtcgttttgc
     2101 atggatgact cggaagtctg aagaaacaat taccccatgg aattttgaag aagttgtcga
     2161 taaaggtgct tcagctcaat catttattga acgcatgaca aactttgata aaaatcttcc
     2221 aaatgaaaaa gtactaccaa aacatagttt gctttatgag tattttacgg tttataacga
     2281 attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa ccagcatttc tttcaggtga
     2341 acagaagaaa gccattgttg atttactctt caaaacaaat cgaaaagtaa ccgttaagca
     2401 attaaaagaa gattatttca aaaaaataga atgttttgat agtgttgaaa tttcaggagt
     2461 tgaagataga tttaatgctt cattaggtac ctaccatgat ttgctaaaaa ttattaaaga
     2521 taaagatttt ttggataatg aagaaaatga agatatctta gaggatattg ttttaacatt
     2581 gaccttattt gaagataggg agatgattga ggaaagactt aaaacatatg ctcacctctt
     2641 tgatgataag gtgatgaaac agcttaaacg tcgccgttat actggttggg gacgtttgtc
     2701 tcgaaaattg attaatggta ttagggataa gcaatctggc aaaacaatat tagatttttt
     2761 gaaatcagat ggttttgcca atcgcaattt tatgcagctg atccatgatg atagtttgac
     2821 atttaaagaa gacattcaaa aagcacaagt gtctggacaa ggcgatagtt tacatgaaca
     2881 tattgcaaat ttagctggta gccctgctat taaaaaaggt attttacaga ctgtaaaagt
     2941 tgttgatgaa ttggtcaaag taatggggcg gcataagcca gaaaatatcg ttattgaaat
     3001 ggcacgtgaa aatcagacaa ctcaaaaggg ccagaaaaat tcgcgagagc gtatgaaacg
     3061 aatcgaagaa ggtatcaaag aattaggaag tcagattctt aaagagcatc ctgttgaaaa
     3121 tactcaattg caaaatgaaa agctctatct ctattatctc caaaatggaa gagacatgta
     3181 tgtggaccaa gaattagata ttaatcgttt aagtgattat gatgtcgatc acattgttcc
     3241 acaaagtttc cttaaagacg attcaataga caataaggtc ttaacgcgtt ctgataaaaa
     3301 tcgtggtaaa tcggataacg ttccaagtga agaagtagtc aaaaagatga aaaactattg
     3361 gagacaactt ctaaacgcca agttaatcac tcaacgtaag tttgataatt taacgaaagc
     3421 tgaacgtgga ggtttgagtg aacttgataa agctggtttt atcaaacgcc aattggttga
     3481 aactcgccaa atcactaagc atgtggcaca aattttggat agtcgcatga atactaaata
     3541 cgatgaaaat gataaactta ttcgagaggt taaagtgatt accttaaaat ctaaattagt
     3601 ttctgacttc cgaaaagatt tccaattcta taaagtacgt gagattaaca attaccatca
     3661 tgcccatgat gcgtatctaa atgccgtcgt tggaactgct ttgattaaga aatatccaaa
     3721 acttgaatcg gagtttgtct atggtgatta taaagtttat gatgttcgta aaatgattgc
     3781 taagtctgag caagaaatag gcaaagcaac cgcaaaatat ttcttttact ctaatatcat
     3841 gaacttcttc aaaacagaaa ttacacttgc aaatggagag attcgcaaac gccctctaat
     3901 cgaaactaat ggggaaactg gagaaattgt ctgggataaa gggcgagatt ttgccacagt
     3961 gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag aaaacagaag tacagacagg
     4021 cggattctcc aaggagtcaa ttttaccaaa aagaaattcg gacaagctta ttgctcgtaa
     4081 aaaagactgg gatccaaaaa aatatggtgg ttttgatagt ccaacggtag cttattcagt
     4141 cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag ttaaaatccg ttaaagagtt
     4201 actagggatc acaattatgg aaagaagttc ctttgaaaaa aatccgattg actttttaga
     4261 agctaaagga tataaggaag ttaaaaaaga cttaatcatt aaactaccta aatatagtct
     4321 ttttgagtta gaaaacggtc gtaaacggat gctggctagt gccggagaat tacaaaaagg
     4381 aaatgagctg gctctgccaa gcaaatatgt gaatttttta tatttagcta gtcattatga
     4441 aaagttgaag ggtagtccag aagataacga acaaaaacaa ttgtttgtgg agcagcataa
     4501 gcattattta gatgagatta ttgagcaaat cagtgaattt tctaagcgtg ttattttagc
     4561 agatgccaat ttagataaag ttcttagtgc atataacaaa catagagaca aaccaatacg
     4621 tgaacaagca gaaaatatta ttcatttatt tacgttgacg aatcttggag ctcccgctgc
     4681 ttttaaatat tttgatacaa caattgatcg taaacgatat acgtctacaa aagaagtttt
     4741 agatgccact cttatccatc aatccatcac tggtctttat gaaacacgca ttgatttgag
     4801 tcagctagga ggtgacccca agaagaagag gaaggtgatg gataagcatc accaccacca
     4861 tcactaatag gtaatctctg cttaaaagca cagaatctaa gatccctgcc atttggcggg
     4921 gattttttta tttgttttca ggaaataaat aatcgatcgc gtaataaaat ctattattat
     4981 ttttgtgaag aataaatttg ggtgcaatga gaatgcgcag gccctttcgt ctcgcgcgtt
     5041 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     5101 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     5161 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatagtc
     5221 gactaatacg actcactata ggtgacatca attattatac atgttttaga gctagaaata
     5281 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     5341 agcatccaaa ctcgagtaag gatctccagg catcaaataa aacgaaaggc tcagtcgaaa
     5401 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcgtcga cactatggtt
     5461 gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     5521 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5581 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5641 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5701 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5761 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5821 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5881 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5941 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     6001 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6061 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6121 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6181 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6241 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6301 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6361 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6421 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6481 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6541 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6601 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6661 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6721 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6781 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6841 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6901 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6961 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     7021 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7081 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7141 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7201 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7261 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7321 ccttttgctc acatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     7381 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     7441 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     7501 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     7561 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     7621 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     7681 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     7741 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     7801 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     7861 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     7921 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     7981 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     8041 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     8101 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     8161 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     8221 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     8281 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     8341 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     8401 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     8461 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     8521 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     8581 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tg
//
