LOCUS       sequence-275425-        8451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            SpCas9-HF1(N497A/R661A/Q695A/Q926A) with a c-terminal bi-partite 
            NLS, 3x flag tag, and P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8451)
  AUTHORS   Walton RT, Christie KA, Whittaker MN, Kleinstiver BP
  TITLE     Unconstrained genome targeting with near-PAMless engineered 
            CRISPR-Cas9 variants.
  JOURNAL   Science. 2020 Apr 17;368(6488):290-296. doi: 
            10.1126/science.aba8853. Epub 2020 Mar 26.
  PUBMED    32217751
REFERENCE   2  (bases 1 to 8451)
  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: 
            "10.1126/science.aba8853"; journalName: "Science"; date: 
            "2020-04-17- 17"; volume: "368"; issue: "6488"; pages: "290-296"
COMMENT     Sequence Label: pCMV-T7-SpCas9-HF1-P2A-EGFP (RTW3505)
FEATURES             Location/Qualifiers
     source          1..8451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        95..474
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        475..678
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     629..649
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     720..739
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        720..738
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      746..755
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(752..2239,2240..2242,2243..2731,2732..2734,2735..2833,
                     2834..2836,2837..3526,3527..3529,3530..4855)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             752..4855
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4910..4930
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4943..5008
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5027..5083
                     /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             join(5084..5086,5087..5089,5090..5803)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5084..5086,5087..5089,5090..5800)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5390..5409)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5737..5758
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5809..5826
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5849..5866)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5855..6079
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6150..6166)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6150..6166)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6163..6185)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6174..6190
                     /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(6198..6204,6205..6222,6223..6228))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6243..6264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6381..6398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6552..7140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6632..6651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7311..8102,8103..8171))
                     /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     7934..7953
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8172..8276)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8355..8374)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
       61 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
      121 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      181 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      241 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      301 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      361 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      421 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      481 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      541 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      601 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      661 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc cgcggccgct
      721 aatacgactc actataggga gagccgccac catggacaag aagtacagca tcggcctgga
      781 catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa
      841 gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc
      901 cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag
      961 aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat
     1021 ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga
     1081 taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga
     1141 gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga
     1201 cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat
     1261 cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca
     1321 gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc
     1381 catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc
     1441 cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc
     1501 caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac
     1561 ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt
     1621 tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac
     1681 cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca
     1741 ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat
     1801 tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga
     1861 agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct
     1921 cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat
     1981 cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta
     2041 cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta
     2101 ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga
     2161 ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag
     2221 cttcatcgag cggatgaccg ccttcgataa gaacctgccc aacgagaagg tgctgcccaa
     2281 gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt
     2341 gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga
     2401 cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa
     2461 gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc
     2521 cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga
     2581 ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga
     2641 gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca
     2701 gctgaagcgg cggagataca ccggctgggg cgccctgagc cggaagctga tcaacggcat
     2761 ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa
     2821 cagaaacttc atggccctga tccacgacga cagcctgacc tttaaagagg acatccagaa
     2881 agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag
     2941 ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt
     3001 gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac
     3061 ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga
     3121 gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa
     3181 gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat
     3241 caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga
     3301 ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt
     3361 gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa
     3421 gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga
     3481 actggataag gccggcttca tcaagagaca gctggtggaa acccgggcca tcacaaagca
     3541 cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat
     3601 ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt
     3661 ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa
     3721 cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta
     3781 cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg
     3841 caaggctacc gccaagtact tcttctacag caacatcatg aactttttca agaccgagat
     3901 taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg
     3961 ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc
     4021 ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat
     4081 cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa
     4141 gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga
     4201 aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga
     4261 aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt
     4321 gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg
     4381 gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc
     4441 caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga
     4501 ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat
     4561 cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt
     4621 gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat
     4681 ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac
     4741 catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca
     4801 gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacggatc
     4861 cggcggaggc ggaagcggga aaagaaccgc cgacggcagc gaattcgagc ccaagaagaa
     4921 gaggaaagtc tcgagcggag gcgactacaa agaccatgac ggtgattata aagatcatga
     4981 catcgattac aaggatgacg atgacaaggg aggcggtgga agcggcgcaa caaacttctc
     5041 tctgctgaaa caagccggag atgtcgaaga gaatcctgga ccgatggtga gcaagggcga
     5101 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     5161 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     5221 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     5281 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     5341 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     5401 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     5461 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     5521 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     5581 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     5641 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     5701 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     5761 cgccgccggg atcactctcg gcatggacga gctgtacaag tgaccggtca tcatcaccat
     5821 caccattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     5881 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5941 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     6001 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     6061 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg
     6121 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     6181 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc
     6241 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6301 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6361 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     6421 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     6481 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     6541 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     6601 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6661 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6721 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6781 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6841 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6901 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6961 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     7021 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     7081 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     7141 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     7201 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     7261 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     7321 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     7381 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     7441 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     7501 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     7561 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     7621 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     7681 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     7741 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7801 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7861 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7921 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7981 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     8041 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     8101 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     8161 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     8221 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     8281 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta
     8341 gggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     8401 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac a
//
