LOCUS       Exported                9220 bp ds-DNA     circular SYN 29-NOV-2024
DEFINITION  Cas9 from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA 
            (V2.0).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9220)
  AUTHORS   Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F
  TITLE     Genome engineering using the CRISPR-Cas9 system.
  JOURNAL   Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. 
            Epub 2013 Oct 24.
  PUBMED    24157548
REFERENCE   2  (bases 1 to 9220)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Dec 11, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9220
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1051..1278
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1290..1299
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1299..1364
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1371..1391
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1416..5516
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1416..5516
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5517..5564
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5580..5633
                     /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             5634..6233
                     /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(5634..5653)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6130..6150
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6261..6278)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6267..6474
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6483..6623
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6483..6612
                     /label=AAV2 ITR
     rep_origin      6698..7153
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6785..6804)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6995..7016
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7170..7189)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7289..7311
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7349..7367)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7435..7539
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7540..8400
                     /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(7758..7777)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8571..9159
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9060..9079
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg aattcccaaa tgaagatggc gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg cggcgccccg ggtggggggg gggggggggg gggggggggg gggggggggg
      901 cggggggggg tggggggggg cggggggggg ggggggggga ggcggagagg tgcggcggca
      961 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg
     1021 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg
     1081 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
     1141 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag caagaggtaa
     1201 gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga gcacctgcct
     1261 gaaatcactt tttttcaggt tggaccggtg ccaccatgga ctataaggac cacgacggag
     1321 actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga
     1381 agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg
     1441 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
     1501 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
     1561 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
     1621 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
     1681 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
     1741 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
     1801 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     1861 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     1921 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
     1981 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     2041 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     2101 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc
     2161 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     2221 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     2281 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     2341 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     2401 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     2461 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
     2521 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     2581 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     2641 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     2701 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     2761 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     2821 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
     2881 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     2941 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
     3001 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
     3061 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     3121 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct
     3181 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg
     3241 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag
     3301 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc
     3361 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca
     3421 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca
     3481 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga
     3541 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca
     3601 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag
     3661 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca
     3721 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag
     3781 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga
     3841 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca
     3901 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg
     3961 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg
     4021 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca
     4081 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg
     4141 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc
     4201 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga
     4261 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt
     4321 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga
     4381 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt
     4441 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg
     4501 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga
     4561 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg
     4621 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc
     4681 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta
     4741 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga
     4801 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg
     4861 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg
     4921 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag
     4981 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc
     5041 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct
     5101 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg
     5161 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca
     5221 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag
     5281 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca
     5341 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca
     5401 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc
     5461 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa
     5521 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaaggaattc ggcagtggag
     5581 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccaatgaccg
     5641 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     5701 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     5761 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     5821 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     5881 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     5941 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     6001 ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     6061 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     6121 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     6181 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgagaattct
     6241 aactagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     6301 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     6361 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     6421 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
     6481 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     6541 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     6601 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     6661 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     6721 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     6781 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     6841 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     6901 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     6961 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     7021 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     7081 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     7141 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     7201 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     7261 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     7321 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     7381 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     7441 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     7501 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     7561 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     7621 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     7681 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     7741 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     7801 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     7861 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     7921 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     7981 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     8041 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     8101 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     8161 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     8221 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     8281 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     8341 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     8401 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     8461 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     8521 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     8581 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     8641 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     8701 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     8761 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     8821 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     8881 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     8941 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     9001 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     9061 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     9121 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     9181 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
