LOCUS       sequence-274763-       12838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized high-fidelity SpCas9 and blasticidin
            resistance: EFS promoter-SpCas9-HF1-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: SpCas9-HF1
FEATURES             Location/Qualifiers
     source          1..12838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    84..201
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        294..505
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      524..533
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(530..2017,2018..2020,2021..2509,2510..2512,2513..2611,
                     2612..2614,2615..3304,3305..3307,3308..4633)
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             530..4633
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4634..4681
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4682..4705
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4715..4771
                     /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             4772..5170
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5195..5783
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5248..5268)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5666..5677)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5786..5802)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5787..5803)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6308..6488
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6514..6531)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6520..6744
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6790..7218
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6877..6896)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7087..7108
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7227..7247)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7392..7526
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7453..7472
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7588..7635
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7654..8028
                     /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    8158..8279
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8195..8214)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8249..8268
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8328..8344)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8328..8344)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8341..8363)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8352..8368
                     /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(8376..8382,8383..8400,8401..8406))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8421..8442
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8559..8576)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8730..9318)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8810..8829)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9489..10280,10281..10349))
                     /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     10112..10131
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10350..10454)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10529..10548)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10720..11099
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11101..11299
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11254..11274
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11317..11497
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11544..11669
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12162..12395
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12580..12624
                     /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 acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca attctgcaga
       61 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
      121 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
      181 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggtta
      241 attagctagc taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga
      301 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc
      361 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
      421 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
      481 caacgggttt gccgccagaa cacaggaccg gttctagagc gctgccacca tggacaagaa
      541 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
      601 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
      661 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
      721 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
      781 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
      841 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
      901 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
      961 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     1021 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     1081 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     1141 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     1201 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     1261 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     1321 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     1381 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     1441 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     1501 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     1561 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     1621 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     1681 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     1741 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     1801 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     1861 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     1921 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     1981 gggcgcttcc gcccagagct tcatcgagcg gatgaccgcc ttcgataaga acctgcccaa
     2041 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     2101 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     2161 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     2221 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     2281 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     2341 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     2401 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     2461 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggcg ccctgagccg
     2521 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     2581 gtccgacggc ttcgccaaca gaaacttcat ggccctgatc cacgacgaca gcctgacctt
     2641 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     2701 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     2761 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     2821 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     2881 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     2941 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     3001 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     3061 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     3121 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     3181 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     3241 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     3301 ccgggccatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     3361 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     3421 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     3481 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     3541 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     3601 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     3661 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     3721 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     3781 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     3841 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     3901 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     3961 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctact
     4021 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     4081 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     4141 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     4201 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     4261 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     4321 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     4381 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     4441 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     4501 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     4561 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     4621 gctgggaggc gacaagcgac ctgccgccac aaagaaggct ggacaggcta agaagaagaa
     4681 agattacaaa gacgatgacg ataagggatc cggcgcaaca aacttctctc tgctgaaaca
     4741 agccggagat gtcgaagaga atcctggacc gatggccaag cctttgtctc aagaagaatc
     4801 caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag
     4861 cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca
     4921 ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc
     4981 tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg
     5041 cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca tagtgaagga
     5101 cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg
     5161 ggagggctaa gaattcgata tcaagcttat cggtaatcaa cctctggatt acaaaatttg
     5221 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     5281 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     5341 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     5401 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     5461 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     5521 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     5581 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     5641 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     5701 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     5761 ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgagacctag aaaaacatgg
     5821 agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca
     5881 agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta
     5941 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     6001 tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt
     6061 ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga cctttggatg
     6121 gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa
     6181 cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt
     6241 agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga
     6301 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     6361 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     6421 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     6481 ctctagcagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     6541 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     6601 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     6661 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     6721 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     6781 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     6841 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     6901 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     6961 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     7021 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     7081 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     7141 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     7201 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     7261 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     7321 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     7381 agcaaccata gtcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     7441 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     7501 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     7561 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
     7621 atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg
     7681 gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc
     7741 tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc
     7801 atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc
     7861 ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc
     7921 tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc
     7981 gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga
     8041 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     8101 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     8161 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     8221 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     8281 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     8341 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     8401 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     8461 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     8521 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     8581 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     8641 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     8701 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     8761 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     8821 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     8881 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     8941 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     9001 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     9061 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     9121 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     9181 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     9241 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     9301 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     9361 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     9421 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     9481 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     9541 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     9601 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     9661 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     9721 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     9781 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     9841 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     9901 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     9961 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
    10021 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
    10081 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
    10141 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
    10201 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
    10261 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
    10321 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
    10381 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
    10441 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
    10501 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
    10561 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
    10621 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
    10681 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
    10741 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
    10801 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
    10861 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
    10921 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
    10981 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
    11041 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
    11101 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
    11161 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
    11221 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
    11281 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
    11341 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
    11401 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
    11461 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
    11521 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
    11581 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
    11641 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
    11701 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
    11761 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
    11821 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
    11881 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
    11941 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
    12001 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
    12061 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
    12121 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
    12181 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
    12241 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
    12301 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
    12361 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
    12421 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
    12481 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
    12541 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
    12601 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
    12661 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
    12721 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
    12781 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagag
//
