LOCUS       sequence-274774-       12838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized evolved SpCas9 and blasticidin 
            resistance: EFS promoter-evoCas9-NLS-FLAG-P2A-BSD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12838)
  AUTHORS   Kim N, Kim HK, Lee S, Seo JH, Choi JW, Park J, Min S, Yoon S, Cho 
            SR, Kim HH
  TITLE     Prediction of the sequence-specific cleavage activity of Cas9 
            variants.
  JOURNAL   Nat Biotechnol. 2020 Nov;38(11):1328-1336. doi: 
            10.1038/s41587-020-0537-9. Epub 2020 Jun 8.
  PUBMED    32514125
REFERENCE   2  (bases 1 to 12838)
  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"; doi: 
            "10.1038/s41587-020-0537-9"; journalName: "Nat Biotechnol"; date: 
            "2020-11"; volume: "38"; issue: "11"; pages: "1328-1336"
COMMENT     Sequence Label: evoCas9
FEATURES             Location/Qualifiers
     source          1..12838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    158..275
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        368..579
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      598..607
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             604..4707
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 variant with improved
                     specificity (Casini et al., 2018)"
                     /label=evoCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERVTNFDKNLPNEKVLPKHSLLNEY
                     FTVYNELTEVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGQLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4708..4755
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4756..4779
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4789..4845
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4846..5244
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5269..5857
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5322..5342)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5740..5751)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5860..5876)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5861..5877)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6382..6562
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6588..6605)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6594..6818
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6864..7292
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6951..6970)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7161..7182
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7301..7321)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7466..7600
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7527..7546
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7662..7709
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7728..8102
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    8232..8353
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8269..8288)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8323..8342
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8402..8418)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8402..8418)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8415..8437)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8426..8442
                     /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)."
     promoter        complement(join(8450..8456,8457..8474,8475..8480))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8495..8516
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8633..8650)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8804..9392)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8884..8903)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9563..10354,10355..10423))
                     /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     10186..10205
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10424..10528)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10603..10622)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10794..11173
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11175..11373
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11328..11348
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11391..11571
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11618..11743
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12236..12469
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12654..12698
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
ORIGIN
        1 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
       61 aggtggagag agagacagag acagatccat tcgattagtg aacggatcgg cactgcgtgc
      121 gccaattctg cagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg
      181 gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag
      241 aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag
      301 atccagtttg gttaattagc tagctaggtc ttgaaaggag tgggaattgg ctccggtgcc
      361 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
      421 aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
      481 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
      541 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggttcta gagcgctgcc
      601 accatggaca agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc
      661 gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac
      721 cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc
      781 gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc
      841 tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac
      901 agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc
      961 ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga
     1021 aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc
     1081 cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc
     1141 gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac
     1201 cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc
     1261 agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga
     1321 aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc
     1381 gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg
     1441 gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc
     1501 atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc
     1561 tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg
     1621 cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac
     1681 gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc
     1741 ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg
     1801 cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg
     1861 cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag
     1921 atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac
     1981 agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag
     2041 gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcgggtgac caacttcgat
     2101 aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgaacga gtacttcacc
     2161 gtgtataacg agctgaccga ggtgaaatac gtgaccgagg gaatgagaaa gcccgccttc
     2221 ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg
     2281 accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa
     2341 atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa
     2401 attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc
     2461 gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat
     2521 gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg
     2581 ggccagctga gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc
     2641 ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac
     2701 gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc
     2761 ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag
     2821 acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc
     2881 gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag
     2941 agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac
     3001 cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg
     3061 cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac
     3121 catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga
     3181 agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg
     3241 aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat
     3301 ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga
     3361 cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg
     3421 aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag
     3481 tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac
     3541 aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa
     3601 aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg
     3661 aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac
     3721 agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag
     3781 cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat
     3841 tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag
     3901 gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg
     3961 atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg
     4021 gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt
     4081 gtgaaagagc tactggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc
     4141 gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct
     4201 aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa
     4261 ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc
     4321 agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg
     4381 gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga
     4441 gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat
     4501 aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga
     4561 gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc
     4621 aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg
     4681 atcgacctgt ctcagctggg aggcgacaag cgacctgccg ccacaaagaa ggctggacag
     4741 gctaagaaga agaaagatta caaagacgat gacgataagg gatccggcgc aacaaacttc
     4801 tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccgatggc caagcctttg
     4861 tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc
     4921 tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt
     4981 gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct
     5041 gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc
     5101 atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa
     5161 gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc
     5221 tctggttatg tgtgggaggg ctaagaattc gatatcaagc ttatcggtaa tcaacctctg
     5281 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     5341 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     5401 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     5461 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     5521 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     5581 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     5641 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     5701 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     5761 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     5821 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgagac
     5881 ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc
     5941 tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta
     6001 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     6061 ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac
     6121 acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca
     6181 ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc
     6241 aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg
     6301 gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca catggcccga
     6361 gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     6421 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     6481 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     6541 tcagtgtgga aaatctctag cagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     6601 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     6661 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     6721 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     6781 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     6841 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     6901 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     6961 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     7021 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     7081 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     7141 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     7201 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     7261 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     7321 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     7381 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     7441 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccagttc cgcccattct
     7501 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     7561 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     7621 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg
     7681 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga
     7741 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
     7801 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
     7861 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
     7921 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
     7981 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
     8041 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
     8101 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     8161 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     8221 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     8281 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     8341 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     8401 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     8461 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     8521 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     8581 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     8641 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     8701 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     8761 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     8821 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     8881 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     8941 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     9001 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     9061 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     9121 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     9181 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     9241 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     9301 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     9361 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     9421 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     9481 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     9541 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     9601 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     9661 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     9721 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     9781 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     9841 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     9901 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     9961 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
    10021 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
    10081 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
    10141 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
    10201 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
    10261 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
    10321 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
    10381 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
    10441 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
    10501 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
    10561 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
    10621 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
    10681 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
    10741 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
    10801 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
    10861 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
    10921 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
    10981 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
    11041 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
    11101 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
    11161 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
    11221 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
    11281 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
    11341 taggcgtgta cggtgggagg tctatataag cagcgcgttt tgcctgtact gggtctctct
    11401 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
    11461 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
    11521 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
    11581 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
    11641 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
    11701 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
    11761 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
    11821 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
    11881 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
    11941 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
    12001 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
    12061 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
    12121 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
    12181 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
    12241 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
    12301 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
    12361 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
    12421 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
    12481 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
    12541 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
    12601 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
    12661 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
    12721 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
    12781 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatc
//
