LOCUS       sequence-274798-       12838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized rationally engineered SpCas9 variant
            and blasticidin resistance: EFS promoter-SpCas9-NG-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-NG
FEATURES             Location/Qualifiers
     source          1..12838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    127..244
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        337..548
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      567..576
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             573..4676
                     /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
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4677..4724
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4725..4748
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4758..4814
                     /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             4815..5213
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5238..5826
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5291..5311)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5709..5720)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5829..5845)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5830..5846)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6351..6531
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6557..6574)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6563..6787
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6833..7261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6920..6939)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7130..7151
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7270..7290)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7435..7569
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7496..7515
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7631..7678
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7697..8071
                     /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    8201..8322
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8238..8257)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8292..8311
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8371..8387)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8371..8387)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8384..8406)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8395..8411
                     /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(8419..8425,8426..8443,8444..8449))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8464..8485
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8602..8619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8773..9361)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8853..8872)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9532..10323,10324..10392))
                     /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     10155..10174
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10393..10497)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10572..10591)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10763..11142
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11144..11342
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11297..11317
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11360..11540
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11587..11712
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12205..12438
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12623..12667
                     /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 cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt
       61 cgattagtga acggatcggc actgcgtgcg ccaattctgc agacaaatgg cagtattcat
      121 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
      181 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
      241 ttttcgggtt tattacaggg acagcagaga tccagtttgg ttaattagct agctaggtct
      301 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
      361 tccccgagaa gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg
      421 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
      481 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
      541 gaacacagga ccggttctag agcgctgcca ccatggacaa gaagtacagc atcggcctgg
      601 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
      661 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
      721 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
      781 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
      841 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
      901 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
      961 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     1021 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     1081 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
     1141 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     1201 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     1261 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc
     1321 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     1381 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     1441 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     1501 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     1561 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     1621 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
     1681 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     1741 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     1801 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     1861 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     1921 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     1981 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
     2041 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     2101 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
     2161 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
     2221 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     2281 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct
     2341 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg
     2401 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag
     2461 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc
     2521 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca
     2581 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca
     2641 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga
     2701 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca
     2761 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag
     2821 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca
     2881 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag
     2941 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga
     3001 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca
     3061 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg
     3121 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg
     3181 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca
     3241 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg
     3301 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc
     3361 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga
     3421 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt
     3481 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga
     3541 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt
     3601 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg
     3661 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga
     3721 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg
     3781 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc
     3841 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta
     3901 tccggcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga
     3961 agtacggcgg cttcgtgagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg
     4021 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct actggggatc accatcatgg
     4081 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag
     4141 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc
     4201 ggaagagaat gctggcctct gcccggttcc tgcagaaggg aaacgaactg gccctgccct
     4261 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg
     4321 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca
     4381 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag
     4441 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca
     4501 tccacctgtt taccctgacc aatctgggag cccctcgggc cttcaagtac tttgacacca
     4561 ccatcgaccg gaaggtgtac cggagcacca aagaggtgct ggacgccacc ctgatccacc
     4621 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaagc
     4681 gacctgccgc cacaaagaag gctggacagg ctaagaagaa gaaagattac aaagacgatg
     4741 acgataaggg atccggcgca acaaacttct ctctgctgaa acaagccgga gatgtcgaag
     4801 agaatcctgg accgatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag
     4861 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc
     4921 tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt
     4981 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta
     5041 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg
     5101 tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga
     5161 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagaattcg
     5221 atatcaagct tatcggtaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     5281 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     5341 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     5401 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     5461 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     5521 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     5581 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     5641 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     5701 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     5761 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     5821 ccccgcatcg ataccgtcga cctcgagacc tagaaaaaca tggagcaatc acaagtagca
     5881 atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg
     5941 gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     6001 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     6061 aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact
     6121 acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac
     6181 cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc
     6241 ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca
     6301 gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct
     6361 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     6421 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     6481 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agggcccgtt
     6541 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     6601 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     6661 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     6721 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     6781 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     6841 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     6901 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     6961 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     7021 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     7081 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     7141 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     7201 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     7261 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     7321 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     7381 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     7441 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     7501 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     7561 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     7621 atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa
     7681 ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
     7741 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
     7801 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
     7861 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
     7921 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
     7981 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
     8041 cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc
     8101 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     8161 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     8221 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     8281 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     8341 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     8401 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     8461 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     8521 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     8581 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     8641 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     8701 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     8761 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     8821 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     8881 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     8941 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     9001 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     9061 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     9121 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     9181 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     9241 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     9301 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     9361 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     9421 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     9481 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     9541 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     9601 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     9661 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     9721 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     9781 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     9841 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     9901 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     9961 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
    10021 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
    10081 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
    10141 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
    10201 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
    10261 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
    10321 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
    10381 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
    10441 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
    10501 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
    10561 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
    10621 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
    10681 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
    10741 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
    10801 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
    10861 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
    10921 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
    10981 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
    11041 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
    11101 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
    11161 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
    11221 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
    11281 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
    11341 agcgcgtttt gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
    11401 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
    11461 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
    11521 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
    11581 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
    11641 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
    11701 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
    11761 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
    11821 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
    11881 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
    11941 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
    12001 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat
    12061 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
    12121 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
    12181 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
    12241 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
    12301 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
    12361 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
    12421 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
    12481 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
    12541 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
    12601 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
    12661 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
    12721 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
    12781 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgagggga
//
