LOCUS       sequence-450820-        9911 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CBh-eSpCas9(1.1)-2A-Puro, and 2X hU6-sgRNA (Sp) with a BbsI golden 
            gate cloning backbone for sgRNA. sgRNA uses 3TC scaffold for 
            increased sgRNA expression. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9911)
  AUTHORS   Chey YCJ, Gierus L, Lushington C, Arudkumar JC, B Geiger A, Staker 
            LG, Robertson LJ, Pfitzner C, Kennedy JG, Lee RHB, Godahewa GI, 
            Adikusuma F, Thomas PQ
  TITLE     Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA 
            transcript levels through scaffold poly-T tract reduction.
  JOURNAL   BMC Genomics. 2025 Feb 12;26(1):138. doi: 
            10.1186/s12864-025-11317-2.
  PUBMED    39939860
REFERENCE   2  (bases 1 to 9911)
  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"; journalName: "BMC 
            Genomics."; date: "2025-02-12"; volume: "26(1):138. doi"; pages: " 
            10.1186/s12864-025-11317-2"
COMMENT     Sequence Label: Pdg459-eSp(1.1) V3
FEATURES             Location/Qualifiers
     source          1..9911
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          375..602
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      614..623
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             623..688
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             695..715
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(740..3277,3278..3280,3281..3742,3743..3745,3746..3913,
                     3914..3916,3917..4840)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4841..4888
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4904..4957
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4958..5557
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4958..4977)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5454..5474
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5585..5602)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5591..5798
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(5876..5951)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5969..6217)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     rep_origin      6300..6755
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6387..6406)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6597..6618
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6772..6791)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6891..6913
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6951..6969)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7037..7141
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7142..7210,7211..8002)
                     /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     complement(7360..7379)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8173..8761
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8662..8681
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8823..9071
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        9090..9165
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        9262..9547
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ccccctgtyw tttgkcccma gtwawtdhmy wtdtgggttt ttcytycttg gcawkyycwt
       61 yttstttttw kyyyccckyt kttcccrkgg gcgrggknsc cccccccttt ctcctycccn
      121 cccccccccc cccccccccc cccccccccn tatttttttt tttttttttt tttttatttg
      181 ttgngggggg gggggggggg gggggggggg ggggggcgcg cgcncgnggc ggggcggggc
      241 ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg
      301 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
      361 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg
      421 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
      481 acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt
      541 tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc
      601 aggttggacc ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga
      661 tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat
      721 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc
      781 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct
      841 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag
      901 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg
      961 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga
     1021 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg
     1081 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat
     1141 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta
     1201 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
     1261 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct
     1321 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag
     1381 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa
     1441 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa
     1501 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct
     1561 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa
     1621 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc
     1681 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct
     1741 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag
     1801 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt
     1861 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag
     1921 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca
     1981 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga
     2041 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct
     2101 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
     2161 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat
     2221 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta
     2281 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag
     2341 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac
     2401 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt
     2461 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
     2521 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
     2581 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
     2641 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
     2701 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
     2761 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     2821 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     2881 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     2941 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     3001 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     3061 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     3121 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     3181 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     3241 ctacgatgtg gaccatatcg tgcctcagag ctttctggcc gacgactcca tcgacaacaa
     3301 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     3361 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     3421 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     3481 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     3541 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     3601 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     3661 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     3721 cgccctgatc aaaaagtacc ctgcgctgga aagcgagttc gtgtacggcg actacaaggt
     3781 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     3841 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     3901 cgagatccgg aaggcgcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     3961 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     4021 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     4081 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     4141 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     4201 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     4261 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     4321 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     4381 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     4441 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     4501 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     4561 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     4621 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     4681 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
     4741 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     4801 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa
     4861 aaaggccggc caggcaaaaa agaaaaagga attcggcagt ggagagggca gaggaagtct
     4921 gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg accgagtaca agcccacggt
     4981 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     5041 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     5101 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     5161 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     5221 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     5281 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     5341 cggagtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     5401 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     5461 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     5521 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagaa ttctaactag agctcgctga
     5581 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     5641 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     5701 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     5761 ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcaatt ggcgcacgcg
     5821 ctaaaaacgg actagcctta ttttaacttg ctatttctag ctctaaaaca aaaaagcacc
     5881 gactcggtgc cactttttca agttgataac ggactagcct tatttcaact tgctatttct
     5941 agctctgaaa cgtcttctcg aagaccccgg tgtttcgtcc tttccacaag atatataaag
     6001 ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata
     6061 attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta
     6121 atatctttgt gtttacagtc aaattaattc caattatctc tctaacagcc ttgtatcgta
     6181 tatgcaaata tgaaggaatc atgggaaata ggccctccct gcaggggcgc ctgatgcggt
     6241 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     6301 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     6361 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     6421 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     6481 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     6541 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     6601 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     6661 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     6721 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     6781 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     6841 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     6901 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     6961 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     7021 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     7081 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     7141 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     7201 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     7261 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     7321 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     7381 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     7441 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     7501 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     7561 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     7621 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     7681 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     7741 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     7801 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag
     7861 ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     7921 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     7981 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     8041 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     8101 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     8161 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     8221 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     8281 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     8341 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     8401 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     8461 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     8521 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     8581 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     8641 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     8701 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     8761 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     8821 gtgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     8881 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     8941 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     9001 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     9061 gacgaaacac cgggtcttcg agaagacctg tttcagagct agaaatagca agttgaaata
     9121 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
     9181 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
     9241 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc
     9301 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
     9361 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     9421 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
     9481 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     9541 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     9601 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggc
     9661 tngtcgatga aatagcgaca actatgagta atatgaacat ggtgtgagat gaaattacgt
     9721 atgtaagaga cagagacgga ggaggtattt gtttgtaata gtttggagaa gggggcggca
     9781 ggcaaagaat cccctatcag agatcggggg gggggtgggg gtgaggaaaa aaaggaggaa
     9841 cgttatgggg ggtttttctt gtgaggggaa gtctatatat attttgtcta tctttatgca
     9901 gagggagggg a
//
