LOCUS       sequence-274797-       12838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized expanded PAM SpCas9 variant and 
            blasticidin resistance: EFS promoter-xCas9-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: xCas9
FEATURES             Location/Qualifiers
     source          1..12838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    104..221
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        314..525
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      544..553
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             550..4653
                     /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
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4654..4701
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4702..4725
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4735..4791
                     /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             4792..5190
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5215..5803
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5268..5288)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5686..5697)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5806..5822)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5807..5823)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6328..6508
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6534..6551)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6540..6764
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6810..7238
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6897..6916)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7107..7128
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7247..7267)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7412..7546
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7473..7492
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7608..7655
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7674..8048
                     /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    8178..8299
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8215..8234)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8269..8288
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8348..8364)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8348..8364)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8361..8383)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8372..8388
                     /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(8396..8402,8403..8420,8421..8426))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8441..8462
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8579..8596)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8750..9338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8830..8849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9509..10300,10301..10369))
                     /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     10132..10151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10370..10474)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10549..10568)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10740..11119
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11121..11319
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11274..11294
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11337..11517
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11564..11689
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12182..12415
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12600..12644
                     /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 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
       61 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
      121 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
      181 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
      241 gcagagatcc agtttggtta attagctagc taggtcttga aaggagtggg aattggctcc
      301 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
      361 gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
      421 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
      481 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggaccg gttctagagc
      541 gctgccacca tggacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
      601 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
      661 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
      721 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
      781 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
      841 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
      901 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
      961 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     1021 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     1081 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     1141 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     1201 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     1261 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     1321 ctggccgagg ataccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     1381 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     1441 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     1501 agcgcctcta tgatcaagct gtacgacgag caccaccagg acctgaccct gctgaaagct
     1561 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     1621 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     1681 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     1741 ctgctgcgga agcagcggac cttcgacaac ggcatcatcc cccaccagat ccacctggga
     1801 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     1861 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     1921 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     1981 ttcgagaaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     2041 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     2101 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     2161 gccttcctga gcggcgacca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     2221 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     2281 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     2341 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     2401 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     2461 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     2521 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     2581 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat ccagctgatc
     2641 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     2701 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     2761 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     2821 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     2881 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     2941 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     3001 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     3061 gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     3121 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     3181 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     3241 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     3301 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     3361 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     3421 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     3481 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     3541 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     3601 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     3661 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     3721 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     3781 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     3841 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     3901 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     3961 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     4021 aagagtgtga aagagctact ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     4081 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     4141 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     4201 ggcgtgctgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     4261 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     4321 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     4381 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     4441 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     4501 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     4561 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     4621 acacggatcg acctgtctca gctgggaggc gacaagcgac ctgccgccac aaagaaggct
     4681 ggacaggcta agaagaagaa agattacaaa gacgatgacg ataagggatc cggcgcaaca
     4741 aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc gatggccaag
     4801 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
     4861 cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
     4921 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
     4981 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
     5041 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
     5101 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
     5161 ctgccctctg gttatgtgtg ggagggctaa gaattcgata tcaagcttat cggtaatcaa
     5221 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     5281 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     5341 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     5401 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     5461 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     5521 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     5581 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     5641 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     5701 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     5761 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct
     5821 cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat
     5881 tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta
     5941 cctttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     6001 gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc
     6061 taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga
     6121 tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa
     6181 gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat
     6241 gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg
     6301 gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg
     6361 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     6421 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     6481 ttttagtcag tgtggaaaat ctctagcagg gcccgtttaa acccgctgat cagcctcgac
     6541 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     6601 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     6661 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     6721 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     6781 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     6841 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     6901 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     6961 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     7021 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     7081 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     7141 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     7201 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     7261 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     7321 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     7381 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc cagttccgcc
     7441 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     7501 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     7561 aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat
     7621 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca
     7681 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct
     7741 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc
     7801 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc
     7861 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt
     7921 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg
     7981 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc
     8041 aggactgaca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     8101 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     8161 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     8221 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     8281 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     8341 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     8401 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     8461 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     8521 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     8581 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     8641 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     8701 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     8761 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     8821 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     8881 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     8941 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     9001 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     9061 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     9121 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     9181 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     9241 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     9301 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     9361 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     9421 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     9481 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     9541 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     9601 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     9661 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     9721 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     9781 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     9841 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     9901 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     9961 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
    10021 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
    10081 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
    10141 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
    10201 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
    10261 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
    10321 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
    10381 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
    10441 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
    10501 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
    10561 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
    10621 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
    10681 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
    10741 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
    10801 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
    10861 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
    10921 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
    10981 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
    11041 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
    11101 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
    11161 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
    11221 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
    11281 gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt
    11341 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
    11401 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
    11461 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
    11521 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
    11581 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
    11641 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
    11701 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
    11761 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
    11821 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
    11881 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
    11941 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
    12001 caaaagtaag accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata
    12061 tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag
    12121 gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa
    12181 taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa
    12241 tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt
    12301 tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc
    12361 agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga
    12421 tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga
    12481 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa
    12541 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa
    12601 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca
    12661 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
    12721 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
    12781 tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa ggaataga
//
