LOCUS       sequence-408412-        9387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SpCas9 Delta-cys E532C and E1027C variant 
            under T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9387)
  AUTHORS   Lim D, Sreekanth V, Cox KJ, Law BK, Wagner BK, Karp JM, Choudhary A
  TITLE     Engineering designer beta cells with a CRISPR-Cas9 conjugation 
            platform.
  JOURNAL   Nat Commun. 2020 Aug 13;11(1):4043. doi: 10.1038/s41467-020-17725-0.
  PUBMED    32792475
REFERENCE   2  (bases 1 to 9387)
  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: "Nat 
            Commun."; date: "2020-08-13"; volume: "11(1):4043. doi"; pages: " 
            10.1038/s41467-020-17725-0"
COMMENT     Sequence Label: pDL28_Cas9-His Delta-cys E532C/E1207C
FEATURES             Location/Qualifiers
     source          1..9387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /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     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3485..3504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3504..3528
                     /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             3543..3565
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3584..3604
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3605..7708
                     /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
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRISYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTCGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIESFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELCNGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7709..7729
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7736..7753
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7806..7824)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7820..7867
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7904..8359
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7991..8010)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8201..8222
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(8451..9266)
                     /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(8451..9266)
                     /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     9174..9193
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     9258..9277
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     3541 attttgttta actttaagaa ggagatatac catggggttt atgcccaaga agaagaggaa
     3601 ggtgatggat aagaaatact caataggctt agatatcggc acaaatagcg tcggatgggc
     3661 ggtgatcact gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga
     3721 ccgccacagt atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc
     3781 ggaagcgact cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat
     3841 tagctatcta caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca
     3901 tcgacttgaa gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt
     3961 tggaaatata gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg
     4021 aaaaaaattg gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc
     4081 gcatatgatt aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag
     4141 tgatgtggac aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa
     4201 ccctattaac gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc
     4261 aagacgatta gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg
     4321 gaatctcatt gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc
     4381 agaagatgct aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt
     4441 ggcgcaaatt ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc
     4501 tattttactt tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc
     4561 ttcaatgatt aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt
     4621 tcgacaacaa cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata
     4681 tgcaggttat attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat
     4741 tttagaaaaa atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct
     4801 gcgcaagcaa cggacctttg acaacggctc tattccccat caaattcact tgggtgagct
     4861 gcatgctatt ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa
     4921 gattgaaaaa atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa
     4981 tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga
     5041 agaagttgtc gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga
     5101 taaaaatctt ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac
     5161 ggtttataac gaattgacaa aggtcaaata tgttacttgc ggaatgcgaa aaccagcatt
     5221 tctttcaggt gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt
     5281 aaccgttaag caattaaaag aagattattt caaaaaaata gaaagctttg atagtgttga
     5341 aatttcagga gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa
     5401 aattattaaa gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat
     5461 tgttttaaca ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata
     5521 tgctcacctc tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg
     5581 gggacgtttg tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat
     5641 attagatttt ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga
     5701 tgatagtttg acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag
     5761 tttacatgaa catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca
     5821 gactgtaaaa gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat
     5881 cgttattgaa atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga
     5941 gcgtatgaaa cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca
     6001 tcctgttgaa aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg
     6061 aagagacatg tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga
     6121 tcacattgtt ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg
     6181 ttctgataaa aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat
     6241 gaaaaactat tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa
     6301 tttaacgaaa gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg
     6361 ccaattggtt gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat
     6421 gaatactaaa tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa
     6481 atctaaatta gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa
     6541 caattaccat catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa
     6601 gaaatatcca aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg
     6661 taaaatgatt gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta
     6721 ctctaatatc atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa
     6781 acgccctcta atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga
     6841 ttttgccaca gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga
     6901 agtacagaca ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct
     6961 tattgctcgt aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt
     7021 agcttattca gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc
     7081 cgttaaagag ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat
     7141 tgacttttta gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc
     7201 taaatatagt ctttttgagt tatgcaacgg tcgtaaacgg atgctggcta gtgccggaga
     7261 attacaaaaa ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc
     7321 tagtcattat gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt
     7381 ggagcagcat aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg
     7441 tgttatttta gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga
     7501 caaaccaata cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg
     7561 agctcccgct gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac
     7621 aaaagaagtt ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg
     7681 cattgatttg agtcagctag gaggtgaccc caagaagaag aggaaggtgc tcgagcacca
     7741 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     7801 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     7861 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     7921 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     7981 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     8041 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     8101 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     8161 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     8221 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     8281 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     8341 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     8401 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     8461 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     8521 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     8581 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     8641 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     8701 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     8761 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     8821 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     8881 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     8941 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     9001 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     9061 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     9121 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     9181 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     9241 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     9301 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     9361 ccccgtagaa aagatcaaag gatcttc
//
