LOCUS       sequence-267670-       12859 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 12859)
  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 12859)
  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..12859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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"
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2650..2861
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2880..2889
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2886..6989
                     /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             6990..7037
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7038..7061
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7071..7127
                     /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             7128..7526
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    7551..8139
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7604..7624)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8022..8033)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8142..8158)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8143..8159)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             8664..8844
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(8870..8887)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8876..9100
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9146..9574
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9233..9252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9443..9464
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9583..9603)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9588..9917
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9768..9903
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9830..9849
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9965..10012
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10031..10405
                     /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    10535..10656
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10572..10591)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10626..10645
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10705..10721)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10705..10721)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10718..10740)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10729..10745
                     /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(10753..10759,10760..10777,10778..10783))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10798..10819
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10936..10953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11107..11695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11187..11206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11866..12657,12658..12726))
                     /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     12489..12508
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12727..12831)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta gctagctagg tcttgaaagg agtgggaatt ggctccggtg
     2641 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     2701 gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     2761 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     2821 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggaccggttc tagagcgctg
     2881 ccaccatgga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     2941 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     3001 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     3061 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     3121 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     3181 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     3241 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     3301 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     3361 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     3421 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     3481 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     3541 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     3601 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     3661 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     3721 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     3781 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     3841 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     3901 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     3961 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     4021 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     4081 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     4141 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     4201 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     4261 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     4321 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcgggtg accaacttcg
     4381 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgaac gagtacttca
     4441 ccgtgtataa cgagctgacc gaggtgaaat acgtgaccga gggaatgaga aagcccgcct
     4501 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     4561 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     4621 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     4681 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     4741 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     4801 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     4861 ggggccagct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     4921 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     4981 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     5041 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     5101 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     5161 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     5221 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     5281 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     5341 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     5401 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     5461 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     5521 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     5581 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     5641 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     5701 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     5761 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     5821 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     5881 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     5941 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     6001 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     6061 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     6121 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     6181 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     6241 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     6301 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     6361 gtgtgaaaga gctactgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     6421 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     6481 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     6541 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     6601 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     6661 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     6721 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     6781 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     6841 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     6901 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     6961 ggatcgacct gtctcagctg ggaggcgaca agcgacctgc cgccacaaag aaggctggac
     7021 aggctaagaa gaagaaagat tacaaagacg atgacgataa gggatccggc gcaacaaact
     7081 tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg gccaagcctt
     7141 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
     7201 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
     7261 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
     7321 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
     7381 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     7441 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     7501 cctctggtta tgtgtgggag ggctaagaat tcgatatcaa gcttatcggt aatcaacctc
     7561 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     7621 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     7681 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     7741 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     7801 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     7861 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     7921 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     7981 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     8041 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     8101 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgag
     8161 acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg
     8221 cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt
     8281 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     8341 gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc
     8401 acacacaagg ctacttccct gattggcaga actacacacc agggccaggg atcagatatc
     8461 cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag
     8521 ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc
     8581 cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc
     8641 gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc
     8701 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     8761 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     8821 agtcagtgtg gaaaatctct agcagggccc gtttaaaccc gctgatcagc ctcgactgtg
     8881 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     8941 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     9001 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     9061 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     9121 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     9181 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     9241 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     9301 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     9361 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     9421 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     9481 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     9541 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     9601 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     9661 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     9721 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     9781 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     9841 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     9901 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc
     9961 acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
    10021 gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
    10081 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
    10141 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
    10201 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
    10261 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
    10321 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
    10381 cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt
    10441 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
    10501 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
    10561 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
    10621 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
    10681 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
    10741 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
    10801 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
    10861 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
    10921 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
    10981 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
    11041 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
    11101 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
    11161 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
    11221 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
    11281 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
    11341 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
    11401 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
    11461 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
    11521 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
    11581 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
    11641 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
    11701 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
    11761 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
    11821 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
    11881 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
    11941 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
    12001 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
    12061 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
    12121 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
    12181 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
    12241 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
    12301 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
    12361 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
    12421 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
    12481 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
    12541 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
    12601 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
    12661 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
    12721 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
    12781 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
    12841 ccgaaaagtg ccacctgac
//
