LOCUS       sequence-304464-        9321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for SpyCas9, puromycin resistance and 
            mouse Rosa26 guide RNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9321)
  AUTHORS   Danner E, Lebedin M, de la Rosa K, Kuhn R
  TITLE     A homology independent sequence replacement strategy in human cells 
            using a CRISPR nuclease.
  JOURNAL   Open Biol. 2021 Jan;11(1):200283. doi: 10.1098/rsob.200283. Epub 
            2021 Jan 27.
  PUBMED    33499763
REFERENCE   2  (bases 1 to 9321)
  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: "Open 
            Biol."; date: "2021-01"; volume: "11(1):200283. doi"; pages: " 
            10.1098/rsob.200283. Epub 2021 Jan 27"
COMMENT     Sequence Label: pCbh-SpyCas9-2A-puro-pA_U6-sgRosa26
FEATURES             Location/Qualifiers
     source          1..9321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          131..358
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      370..379
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             379..444
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             451..471
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             496..4596
                     /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             496..4596
                     /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             4597..4644
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4660..4713
                     /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             4714..5313
                     /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(4714..4733)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5210..5230
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5341..5358)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5347..5554
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5563..5703
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5563..5692
                     /label=AAV2 ITR
     rep_origin      5778..6233
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5865..5884)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6075..6096
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6250..6269)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6369..6391
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6429..6447)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6515..6619
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6620..6688,6689..7480)
                     /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(6838..6857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7651..8239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8140..8159
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8301..8549
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8301..8321
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8472..8491
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8571..8646
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8743..9028
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 cgvggggsgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct
       61 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc
      121 gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc
      181 gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg
      241 cccttctcct ccgggctgta attagctgag caagaggtaa gggtttaagg gatggttggt
      301 tggtggggta ttaatgttta attacctgga gcacctgcct gaaatcactt tttttcaggt
      361 tggaccggtg ccaccatgga ctataaggac cacgacggag actacaagga tcatgatatt
      421 gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac
      481 ggagtcccag cagccgacaa gaagtacagc atcggcctgg acatcggcac caactctgtg
      541 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
      601 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
      661 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
      721 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
      781 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
      841 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
      901 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
      961 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
     1021 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
     1081 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
     1141 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
     1201 ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
     1261 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
     1321 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
     1381 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
     1441 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
     1501 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
     1561 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
     1621 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
     1681 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
     1741 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
     1801 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
     1861 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
     1921 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
     1981 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
     2041 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
     2101 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
     2161 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
     2221 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
     2281 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
     2341 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
     2401 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
     2461 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
     2521 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
     2581 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
     2641 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
     2701 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
     2761 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
     2821 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
     2881 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
     2941 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
     3001 gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg
     3061 ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg
     3121 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
     3181 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
     3241 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
     3301 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
     3361 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
     3421 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
     3481 ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac
     3541 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
     3601 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
     3661 atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
     3721 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
     3781 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
     3841 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
     3901 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
     3961 ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag
     4021 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
     4081 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
     4141 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
     4201 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
     4261 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
     4321 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
     4381 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
     4441 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
     4501 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
     4561 gagacacgga tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag
     4621 gccggccagg caaaaaagaa aaaggaattc ggcagtggag agggcagagg aagtctgcta
     4681 acatgcggtg acgtcgagga gaatcctggc ccaatgaccg agtacaagcc cacggtgcgc
     4741 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     4801 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     4861 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     4921 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     4981 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     5041 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     5101 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     5161 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     5221 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     5281 acctggtgca tgacccgcaa gcccggtgcc tgagaattct aactagagct cgctgatcag
     5341 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     5401 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     5461 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     5521 aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg
     5581 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     5641 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     5701 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     5761 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5821 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     5881 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5941 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     6001 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     6061 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     6121 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     6181 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     6241 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     6301 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     6361 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     6421 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     6481 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     6541 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     6601 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     6661 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     6721 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     6781 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     6841 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     6901 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6961 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     7021 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     7081 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     7141 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     7201 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     7261 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     7321 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     7381 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     7441 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     7501 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     7561 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     7621 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     7681 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     7741 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     7801 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     7861 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     7921 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     7981 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     8041 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     8101 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     8161 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     8221 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     8281 ctggcctttt gctcacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac
     8341 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     8401 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     8461 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     8521 tatatatctt gtggaaagga cgaaacaccg actccagtct ttctagaaga gttttagagc
     8581 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     8641 cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta
     8701 gcgcgtgcgc caattctgca gacaaatggc tctagaggta cccgttacat aacttacggt
     8761 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc
     8821 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     8881 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     8941 gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     9001 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     9061 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     9121 tgcagcgatg ggggcggggg gggggggggg gggcgnsccc agggggggsg gggggggggg
     9181 gggggggggg gggggggggg gggggggggg gggggggggg sgrgnggggg gggggggggg
     9241 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
     9301 gggggggggg gggggggggg g
//
