LOCUS       sequence-410602-       10752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of increased fidelity SpCas9-HF1 in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10752)
  AUTHORS   Kulcsar PI, Talas A, Toth E, Nyeste A, Ligeti Z, Welker Z, Welker E
  TITLE     Blackjack mutations improve the on-target activities of increased 
            fidelity variants of SpCas9 with 5'G-extended sgRNAs.
  JOURNAL   Nat Commun. 2020 Mar 6;11(1):1223. doi: 10.1038/s41467-020-15021-5.
  PUBMED    32144253
REFERENCE   2  (bases 1 to 10752)
  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: "2020-03-6"; volume: "11(1):1223. doi"; pages: " 
            10.1038/s41467-020-15021-5"
COMMENT     Sequence Label: pET-FLAG-SpCas9-HF1
FEATURES             Location/Qualifiers
     source          1..10752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1291..1482
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1476..1498)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1584..1723
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1639..1658
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1738..1755)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1909..2497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1989..2008)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2608..2627)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2619..3434
                     /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             2619..3434
                     /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     complement(2692..2711)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3526..3981)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3663..3684)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3875..3894
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4018..4065)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4061..4079
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4132..4149)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     4182..4199
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4227..4274)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(join(4275..5600,5601..5603,5604..6293,6294..6296,
                     6297..6395,6396..6398,6399..6887,6888..6890,6891..8375))
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(8400..8420)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(8427..8492)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(8505..8525)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(8556..9653)
                     /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     complement(8559..8582)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(9657..9674)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             9685..9707
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    9722..9746
                     /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     complement(9746..9765)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9747..9765)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9849..9868)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        10074..10151
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(10152..10752,1..482)
                     /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     complement(10171..10190)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
       61 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      121 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      181 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
      241 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      301 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      361 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
      541 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc
      601 gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc
      661 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
      721 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
      781 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
      841 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
      901 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
      961 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     1021 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     1081 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     1141 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     1201 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     1261 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     1321 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     1381 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     1441 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     1501 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     1561 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     1621 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     1681 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     1741 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1801 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1861 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1921 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1981 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2041 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2101 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2161 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2221 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2281 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2341 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2401 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2461 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2521 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa
     2581 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     2641 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     2701 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     2761 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     2821 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     2881 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     2941 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     3001 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     3061 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     3121 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     3181 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     3241 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     3301 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     3361 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     3421 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3481 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     3541 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3601 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3661 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3721 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3781 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3841 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3901 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3961 atgcgccgct acagggcgcg tcccattcgc caatccggat atagttcctc ctttcagcaa
     4021 aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt gctcagcggt
     4081 ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatctc agtggtggtg
     4141 gtggtggtgc tcgagtgcgg ccgcaacaga tggctggcaa ctagaaggca cagtcgaggc
     4201 tgatcagcga gctctaggaa ttcttacttt ttcttttttg cctggccggc ctttttcgtg
     4261 gccgccggcc ttttgtcacc cccaagctgt gacaaatcta tccgagtttc atataatccc
     4321 gtgatggatt ggtgaatcag tgtcgcgtct agcacctcct tggtagaagt gtatcgtttg
     4381 cgatctatcg ttgtgtcaaa atacttgaat gcggctggag cgccgaggtt ggtaagagta
     4441 aacaaatgga taatattttc cgcctgctca cgtatgggtt tatccctgtg cttgttgtat
     4501 gcgcttaata ctttgtccag attggcatca gctaggatga ctctcttact gaattccgaa
     4561 atttgctcta tgatttcgtc gagataatgt ttgtgctgct caacaaaaag ttgcttctgt
     4621 tcgttatctt caggtgaacc tttcaacttc tcgtaatggg acgctaaata caggaaattc
     4681 acgtatttag acggtagtgc gagttcgttc cccttttgaa gctctccggc gctagccaac
     4741 atccgttttc ggccattttc taactcaaac agactatact ttggtagttt aattatgaga
     4801 tcctttttta cttccttgta acctttcgcc tcaaggaagt cgatggggtt cttttcaaaa
     4861 gacgagcgct ccataatcgt tatccccaat aattctttga ctgacttcag tttcttggat
     4921 tttcccttct caacttttgc cactactagg acagaatagg caactgtagg gctatcgaag
     4981 ccaccgtact ttttcgggtc ccagtccttt ttacgagcga tgagcttatc actattcctt
     5041 tttggaagaa tcgattcctt tgaaaaccct ccggtctgca cctcagtttt ctttactatg
     5101 ttgacttggg gcatggacaa aacttttctc accgtcgcga agtcccggcc cttatcccat
     5161 acgatttcac ctgtctcccc attggtttca attaaaggtc gtttgcgtat ctctccgttt
     5221 gccagagtga tttccgtctt aaagaaattc ataatgttag aataaaagaa gtatttggct
     5281 gtagccttgc ctatctcctg ttcgcttttc gcgatcatct tacggacgtc ataaactttg
     5341 taatcaccat acacaaactc actttctagc ttcgggtatt tcttaatgag tgcggtccct
     5401 acgacggcat taagataagc gtcgtgcgca tggtggtagt tatttatctc cctaacttta
     5461 tagaattgaa aatcctttct gaagtccgac accaattttg actttaaagt gattactttg
     5521 acttcccgaa tcagcttatc gttctcgtcg tatttcgtat tcattcggga atctagtatc
     5581 tgcgcaacat gctttgtgat ggcgcgggtt tccacgagct gacgtttaat aaatccggcc
     5641 ttgtcaagtt cagacaagcc acccctctca gctttagtta agttatcgaa ctttctttgc
     5701 gttatcagtt tcgcatttag gagctgccgc caatagttct tcattttctt tacgacttcc
     5761 tcgcttggaa cattgtcact tttccctcgg ttcttatccg agcgtgtaag cactttattg
     5821 tcgattgaat cgtccttcaa aaaggattgg ggtacaatgt gatcgacgtc gtaatcagat
     5881 aaacggttta tgtccagttc ctgatcaaca tacatgtccc ttccattttg taggtaatag
     5941 aggtaaagtt tctcgttctg caattgggta ttttccacag gatgctcctt taagatctgg
     6001 ctgcccagtt ctttaatacc ctcttctatt ctcttcatcc gctctcgact gtttttttgc
     6061 cccttctgag tcgtttgatt ttcgcgtgcc atctcgatta caatgttttc cggtttgtga
     6121 cgtcccatga ccttaactag ctcatccact actttgactg tctggagtat gccctttttg
     6181 atggctggcg aaccagcaag attcgcaata tgttcgtgca atgagtcccc ttgtccggaa
     6241 acctgtgcct tttgtatatc ctctttgaag gttaaagagt catcatggat cagggccata
     6301 aagttcctat tggcgaagcc gtcgctcttt agaaaatcga gaatagtttt accactttgc
     6361 ttgtctctta tcccgttgat aagtttccgc gacaaggctc cccagcccgt atagcgacgc
     6421 ctctttaact gtttcataac cttatcgtcg aacaggtgag cgtatgtttt tagtctttcc
     6481 tcaatcattt cccgatcttc aaagagggta agagtcaaca ctatatcttc taagatatct
     6541 tcattctctt cgttatccag gaagtcctta tctttaatta tctttaggag gtcatgatac
     6601 gtaccaagtg acgcattaaa tcgatcttct accccggaga tctcgacaga atcgaagcat
     6661 tcaattttct taaagtagtc ctctttcaat tgcttaactg tcactttgcg gttggtcttg
     6721 aataacagat ctactattgc tttcttctgt tctccgctta gaaaggcggg tttacgcatg
     6781 ccctcagtga catacttaac tttcgtgagt tcattgtaca ctgtgaaata ctcgtaaagt
     6841 aaactgtgct taggcaatac tttttcgttc ggtaaattct tgtcaaaggc ggtcatcctc
     6901 tcgatgaacg attgagctga cgcaccttta tcgacaactt cctcaaaatt ccagggagta
     6961 atcgtttctt cggactttct tgtcatccag gcgaatctgc tgtttcccct ggccagaggg
     7021 cccacgtagt aggggatgcg gaaggtcagg atcttctcga tcttttcccg gttgtccttc
     7081 aggaatgggt aaaaatcttc ctgccgccgc agaatggcgt gcagctctcc caggtggatc
     7141 tggtggggga tgctgccgtt gtcgaaggtc cgctgcttcc gcagcaggtc ctctctgttc
     7201 agcttcacga gcagttcctc ggtgccgtcc atcttttcca ggatgggctt gatgaacttg
     7261 tagaactctt cctggctggc tccgccgtca atgtagccgg cgtagccgtt cttgctctgg
     7321 tcgaagaaaa tctctttgta cttctcaggc agctgctgcc gcacgagagc tttcagcagg
     7381 gtcaggtcct ggtggtgctc gtcgtatctc ttgatcatag aggcgctcag gggggccttg
     7441 gtgatctcgg tgttcactct caggatgtcg ctcagcagga tggcgtcgga caggttcttg
     7501 gcggccagaa acaggtcggc gtactggtcg ccgatctggg ccagcaggtt gtccaggtcg
     7561 tcgtcgtagg tgtccttgct cagctgcagt ttggcatcct cggccaggtc gaagttgctc
     7621 ttgaagttgg gggtcaggcc caggctcagg gcaatcaggt ttccgaacag gccattcttc
     7681 ttctcgccgg gcagctgggc gatcagattt tccagccgtc tgctcttgct cagtctggca
     7741 gacaggatgg ccttggcgtc cacgccgctg gcgttgatgg ggttttcctc gaacagctgg
     7801 ttgtaggtct gcaccagctg gatgaacagc ttgtccacgt cgctgttgtc ggggttcagg
     7861 tcgccctcga tcaggaagtg gccccggaac ttgatcatgt gggccagggc cagatagatc
     7921 agccgcaggt cggccttgtc ggtgctgtcc accagtttct ttctcaggtg gtagatggtg
     7981 gggtacttct cgtggtaggc cacctcgtcc acgatgttgc cgaagatggg gtgccgctcg
     8041 tgcttcttat cctcttccac caggaaggac tcttccagtc tgtggaagaa gctgtcgtcc
     8101 accttggcca tctcgttgct gaagatctct tgcagatagc agatccggtt cttccgtctg
     8161 gtgtatcttc ttctggcggt tctcttcagc cgggtggcct cggctgtttc gccgctgtcg
     8221 aacagcaggg ctccgatcag gttcttcttg atgctgtgcc ggtcggtgtt gcccagcacc
     8281 ttgaatttct tgctgggcac cttgtactcg tcggtgatca cggcccagcc cacagagttg
     8341 gtgccgatgt ccaggccgat gctgtacttc ttgtcggctg ctgggactcc gtggataccg
     8401 accttccgct tcttctttgg ggccatctta tcgtcatcgt ctttgtaatc aatatcatga
     8461 tccttgtagt ctccgtcgtg gtccttatag tccatcgagg ctgctccctg gaaatacagg
     8521 ttttcactag tgttgttgtt gttcgagctc gaattagtct gcgcgtcttt cagggcttca
     8581 tcgacagtct gacgaccgct ggcggcgttg atcaccgcag tacgcacggc ataccagaaa
     8641 gcggacatct gcgggatgtt cggcatgatt tcacctttct gggcgttttc catagtggcg
     8701 gcaatacgtg gatctttcgc caactcttcc tcgtaagact tcagcgctac ggcacccagc
     8761 ggtttgtctt tattaaccgc ttccagacct tcatcagtca gcagatagtt ttcgaggaac
     8821 tcttttgcca gctctttgtt cggactggcg gcgttaatac ctgcgctcag cacgccaacg
     8881 aacggtttgg atggttgacc cttgaaggtc ggcagtaccg ttacaccata attcactttg
     8941 ctggtgtcga tgttggacca tgcccacggg ccgttgatgg tcatcgctgt ttcgccttta
     9001 ttaaaggcag cttctgcgat ggagtaatcg gtgtctgcat tcatgtgttt gtttttaatc
     9061 aggtcaacca ggaaggtcag acccgctttc gcgccagcgt tatccacgcc cacgtcttta
     9121 atgtcgtact tgccgttttc atacttgaac gcataacccc cgtcagcagc aatcagcggc
     9181 caggtgaagt acggttcttg caggttgaac atcagcgcgc tcttaccttt cgctttcagt
     9241 tctttatcca gcgccgggat ctcttcccag gtttttggcg ggttcggcag cagatctttg
     9301 ttataaatca gcgataacgc ttcaacagcg atcgggtaag caatcagctt gccgttgtaa
     9361 cgtacggcat cccaggtaaa cggatacagc ttgtcctgga acgctttgtc cggggtgatt
     9421 tcagccaaca ggccagattg agcgtagcca ccaaagcggt cgtgtgccca gaagataatg
     9481 tcagggccat cgccagttgc cgcaacctgt gggaatttct cttccagttt atccggatgc
     9541 tcaacggtga ctttaattcc ggtatctttc tcgaatttct taccgacttc agcgagaccg
     9601 ttatagcctt tatcgccgtt aatccagatt accagtttac cttcttcgat ttttccatgg
     9661 tgatggtgat ggtgcatatg tatatctcct tcttaaagtt aaacaaaatt atttctagag
     9721 gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga
     9781 gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
     9841 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct
     9901 catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg
     9961 cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact
    10021 actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca
    10081 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
    10141 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
    10201 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
    10261 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
    10321 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
    10381 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
    10441 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
    10501 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
    10561 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
    10621 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
    10681 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
    10741 gtctggctgg ct
//
