LOCUS       sequence-274789-       12838 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses human codon-optimized SpCas9-VRQR-HF1 and blasticidin 
            resistance: EFS promoter-VRQR-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 Apr 14, 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: VRQR-HF1 variant
FEATURES             Location/Qualifiers
     source          1..12838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    145..262
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        355..566
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      585..594
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             591..4694
                     /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
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4695..4742
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4743..4766
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4776..4832
                     /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             4833..5231
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5256..5844
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5309..5329)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5727..5738)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5847..5863)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5848..5864)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6369..6549
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6575..6592)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6581..6805
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6851..7279
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6938..6957)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7148..7169
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7288..7308)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7453..7587
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7514..7533
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7649..7696
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7715..8089
                     /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    8219..8340
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8256..8275)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8310..8329
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8389..8405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8389..8405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8402..8424)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8413..8429
                     /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(8437..8443,8444..8461,8462..8467))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8482..8503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8620..8637)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8791..9379)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8871..8890)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9550..10341,10342..10410))
                     /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     10173..10192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10411..10515)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10590..10609)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10781..11160
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11162..11360
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11315..11335
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11378..11558
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11605..11730
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12223..12456
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12641..12685
                     /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 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga
       61 gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag
      121 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
      181 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
      241 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt
      301 aattagctag ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag
      361 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgatccggtg
      421 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
      481 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
      541 gcaacgggtt tgccgccaga acacaggacc ggttctagag cgctgccacc atggacaaga
      601 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg
      661 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
      721 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
      781 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
      841 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
      901 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
      961 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1021 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1081 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1141 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1201 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     1261 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     1321 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     1381 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     1441 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     1501 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     1561 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     1621 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     1681 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     1741 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     1801 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     1861 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     1921 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     1981 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2041 agggcgcttc cgcccagagc ttcatcgagc ggatgaccgc cttcgataag aacctgccca
     2101 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2161 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     2221 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     2281 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     2341 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     2401 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     2461 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     2521 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc gccctgagcc
     2581 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     2641 agtccgacgg cttcgccaac agaaacttca tggccctgat ccacgacgac agcctgacct
     2701 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     2761 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     2821 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     2881 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     2941 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3001 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3061 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     3121 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3181 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     3241 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     3301 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     3361 cccgggccat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     3421 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     3481 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     3541 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     3601 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     3661 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     3721 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     3781 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     3841 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     3901 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     3961 aggactggga ccctaagaag tacggcggct tcgtgagccc caccgtggcc tattctgtgc
     4021 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctac
     4081 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4141 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4201 tcgagctgga aaacggccgg aagagaatgc tggcctctgc ccgggaactg cagaagggaa
     4261 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     4321 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     4381 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     4441 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     4501 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     4561 tcaagtactt tgacaccacc atcgaccgga agcagtaccg gagcaccaaa gaggtgctgg
     4621 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     4681 agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct aagaagaaga
     4741 aagattacaa agacgatgac gataagggat ccggcgcaac aaacttctct ctgctgaaac
     4801 aagccggaga tgtcgaagag aatcctggac cgatggccaa gcctttgtct caagaagaat
     4861 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     4921 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     4981 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     5041 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     5101 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
     5161 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     5221 gggagggcta agaattcgat atcaagctta tcggtaatca acctctggat tacaaaattt
     5281 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     5341 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     5401 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     5461 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     5521 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     5581 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     5641 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     5701 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     5761 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     5821 tctccctttg ggccgcctcc ccgcatcgat accgtcgacc tcgagaccta gaaaaacatg
     5881 gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac
     5941 aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt
     6001 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     6061 ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact
     6121 tccctgattg gcagaactac acaccagggc cagggatcag atatccactg acctttggat
     6181 ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga
     6241 acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat
     6301 tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg
     6361 actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     6421 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     6481 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     6541 tctctagcag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     6601 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     6661 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     6721 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     6781 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     6841 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     6901 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     6961 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     7021 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     7081 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     7141 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     7201 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     7261 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     7321 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     7381 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     7441 cagcaaccat agtcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     7501 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     7561 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     7621 tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg
     7681 catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc
     7741 ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt
     7801 ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt
     7861 catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg
     7921 cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc
     7981 ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg
     8041 cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctacg
     8101 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     8161 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     8221 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     8281 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     8341 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     8401 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     8461 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     8521 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     8581 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     8641 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     8701 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     8761 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     8821 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     8881 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     8941 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     9001 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     9061 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     9121 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     9181 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     9241 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     9301 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     9361 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     9421 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     9481 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     9541 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     9601 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     9661 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     9721 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     9781 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     9841 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     9901 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     9961 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
    10021 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
    10081 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
    10141 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
    10201 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
    10261 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
    10321 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
    10381 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
    10441 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
    10501 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
    10561 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
    10621 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
    10681 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
    10741 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
    10801 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
    10861 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
    10921 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
    10981 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
    11041 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
    11101 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
    11161 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
    11221 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
    11281 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
    11341 tgggaggtct atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat
    11401 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
    11461 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
    11521 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
    11581 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
    11641 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
    11701 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
    11761 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
    11821 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
    11881 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
    11941 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
    12001 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
    12061 cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
    12121 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
    12181 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
    12241 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
    12301 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
    12361 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
    12421 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
    12481 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
    12541 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
    12601 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
    12661 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
    12721 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
    12781 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccac
//
