LOCUS       sequence-274761-       12838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized enhanced specificity SpCas9 and 
            blasticidin resistance: EFS promoter-eSpCas9(1.1)-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: eSpCas9(1.1)
FEATURES             Location/Qualifiers
     source          1..12838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    107..224
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        317..528
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      547..556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(553..3093,3094..3096,3097..3558,3559..3561,3562..3729,
                     3730..3732,3733..4656)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4657..4704
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4705..4728
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4738..4794
                     /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             4795..5193
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5218..5806
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5271..5291)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5689..5700)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5809..5825)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5810..5826)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6331..6511
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6537..6554)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6543..6767
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6813..7241
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6900..6919)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7110..7131
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7250..7270)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7415..7549
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7476..7495
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7611..7658
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7677..8051
                     /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    8181..8302
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8218..8237)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8272..8291
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8351..8367)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8351..8367)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8364..8386)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8375..8391
                     /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(8399..8405,8406..8423,8424..8429))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8444..8465
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8582..8599)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8753..9341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8833..8852)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9512..10303,10304..10372))
                     /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     10135..10154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10373..10477)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10552..10571)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10743..11122
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11124..11322
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11277..11297
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11340..11520
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11567..11692
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12185..12418
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12603..12647
                     /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 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
       61 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
      121 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
      181 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
      241 acagcagaga tccagtttgg ttaattagct agctaggtct tgaaaggagt gggaattggc
      301 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
      361 ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
      421 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
      481 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga ccggttctag
      541 agcgctgcca ccatggacaa gaagtacagc atcggcctgg acatcggcac caactctgtg
      601 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
      661 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
      721 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
      781 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
      841 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
      901 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
      961 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
     1021 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
     1081 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
     1141 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
     1201 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
     1261 ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
     1321 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
     1381 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
     1441 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
     1501 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
     1561 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
     1621 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
     1681 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
     1741 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
     1801 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
     1861 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
     1921 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
     1981 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
     2041 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
     2101 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
     2161 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
     2221 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
     2281 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
     2341 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
     2401 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
     2461 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
     2521 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
     2581 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
     2641 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
     2701 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
     2761 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
     2821 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
     2881 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
     2941 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
     3001 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
     3061 gatgtggacc atatcgtgcc tcagagcttt ctggccgacg actccatcga caacaaggtg
     3121 ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg
     3181 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
     3241 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
     3301 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
     3361 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
     3421 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
     3481 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
     3541 ctgatcaaaa agtaccctgc gctggaaagc gagttcgtgt acggcgacta caaggtgtac
     3601 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
     3661 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
     3721 atccggaagg cgcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
     3781 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
     3841 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
     3901 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
     3961 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
     4021 ctgaagagtg tgaaagagct actggggatc accatcatgg aaagaagcag cttcgagaag
     4081 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
     4141 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
     4201 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
     4261 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
     4321 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
     4381 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
     4441 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
     4501 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
     4561 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
     4621 gagacacgga tcgacctgtc tcagctggga ggcgacaagc gacctgccgc cacaaagaag
     4681 gctggacagg ctaagaagaa gaaagattac aaagacgatg acgataaggg atccggcgca
     4741 acaaacttct ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accgatggcc
     4801 aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc
     4861 atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc
     4921 ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg
     4981 ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag
     5041 aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct
     5101 gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa
     5161 ttgctgccct ctggttatgt gtgggagggc taagaattcg atatcaagct tatcggtaat
     5221 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     5281 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     5341 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     5401 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     5461 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     5521 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     5581 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     5641 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     5701 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     5761 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     5821 cctcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct
     5881 gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt cacacctcag
     5941 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     6001 aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg
     6061 atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc
     6121 agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca agagaaggta
     6181 gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct gcatgggatg
     6241 gatgacccgg agagagaagt attagagtgg aggtttgaca gccgcctagc atttcatcac
     6301 atggcccgag agctgcatcc ggactgtact gggtctctct ggttagacca gatctgagcc
     6361 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     6421 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     6481 cccttttagt cagtgtggaa aatctctagc agggcccgtt taaacccgct gatcagcctc
     6541 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     6601 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     6661 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     6721 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     6781 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     6841 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     6901 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     6961 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     7021 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     7081 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     7141 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     7201 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     7261 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     7321 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     7381 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccagttcc
     7441 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     7501 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     7561 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt
     7621 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg
     7681 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt
     7741 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg
     7801 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca
     7861 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg
     7921 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt
     7981 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg
     8041 agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     8101 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     8161 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     8221 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     8281 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     8341 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     8401 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     8461 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     8521 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     8581 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     8641 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     8701 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     8761 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     8821 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     8881 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     8941 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     9001 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     9061 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     9121 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     9181 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     9241 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     9301 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     9361 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     9421 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     9481 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     9541 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     9601 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     9661 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     9721 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     9781 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     9841 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     9901 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     9961 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
    10021 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
    10081 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
    10141 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
    10201 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
    10261 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
    10321 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
    10381 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
    10441 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
    10501 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
    10561 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
    10621 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
    10681 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
    10741 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
    10801 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
    10861 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
    10921 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
    10981 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
    11041 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
    11101 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
    11161 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
    11221 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
    11281 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg
    11341 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
    11401 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
    11461 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
    11521 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
    11581 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
    11641 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
    11701 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
    11761 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
    11821 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
    11881 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
    11941 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
    12001 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
    12061 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
    12121 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
    12181 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
    12241 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
    12301 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
    12361 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
    12421 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
    12481 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
    12541 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
    12601 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
    12661 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
    12721 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
    12781 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaat
//
