LOCUS       sequence-327807-       10071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral introduction of Cas9 into mammalian cell line coupled to 
            expression of blue fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10071)
  AUTHORS   Caeser R, Di Re M, Krupka JA, Gao J, Lara-Chica M, Dias JML, Cooke 
            SL, Fenner R, Usheva Z, Runge HFP, Beer PA, Eldaly H, Pak HK, Park 
            CS, Vassiliou GS, Huntly BJP, Mupo A, Bashford-Rogers RJM, Hodson DJ
  TITLE     Genetic modification of primary human B cells to model high-grade 
            lymphoma.
  JOURNAL   Nat Commun. 2019 Oct 4;10(1):4543. doi: 10.1038/s41467-019-12494-x.
  PUBMED    31586074
REFERENCE   2  (bases 1 to 10071)
  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: "Nat 
            Commun."; date: "2019-10-4"; volume: "10(1):4543. doi"; pages: " 
            10.1038/s41467-019-12494-x"
COMMENT     Sequence Label: MSCV-Cas9-2A-BFP
FEATURES             Location/Qualifiers
     source          1..10071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    519..860
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             927..1343
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1273..1295
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1311..1327
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1354..1377
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1384..1404
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1429..5529
                     /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             1429..5529
                     /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             5530..5577
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5596..5649
                     /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             5650..6351
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     LTR             6404..6918
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    7080..7096
                     /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(7104..7110,7111..7128,7129..7134))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7149..7170
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7287..7304)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7458..8046)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7538..7557)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8217..9008,9009..9077))
                     /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     8840..8859
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9078..9182)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9250..9268
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(9306..9328)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     9428..9447
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9641..9663
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9791..9810
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(10010..10071,1..455)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
       61 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      121 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      181 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      241 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      301 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      361 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      421 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      481 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      541 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      601 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      661 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      721 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      781 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      841 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      901 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
      961 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1021 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1081 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1141 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1201 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1261 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1321 ccctcactcc ttctctaggc gccggaatta atggattaca aagacgatga cgataagatg
     1381 gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac
     1441 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     1501 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     1561 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     1621 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     1681 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     1741 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     1801 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     1861 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     1921 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     1981 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     2041 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     2101 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     2161 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     2221 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     2281 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     2341 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     2401 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     2461 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     2521 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     2581 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     2641 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     2701 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     2761 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     2821 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     2881 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     2941 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     3001 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     3061 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     3121 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     3181 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     3241 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     3301 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     3361 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     3421 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     3481 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     3541 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     3601 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     3661 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     3721 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     3781 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     3841 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     3901 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     3961 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     4021 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     4081 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     4141 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     4201 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     4261 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     4321 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     4381 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     4441 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     4501 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     4561 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     4621 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     4681 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     4741 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     4801 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     4861 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     4921 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     4981 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     5041 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     5101 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     5161 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     5221 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     5281 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     5341 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     5401 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     5461 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
     5521 ggaggcgaca agcgtcctgc tgctactaag aaagctggtc aagctaagaa aaagaaagga
     5581 tccggcagcg gcggggaagg aaggggctcc ctgctcacct gcggcgacgt ggaagagaac
     5641 cccggaccta tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc
     5701 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     5761 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     5821 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
     5881 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
     5941 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     6001 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
     6061 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
     6121 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat caagaccaca
     6181 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
     6241 agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca
     6301 gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatta actcgagatc
     6361 gacaaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag accccacctg
     6421 taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa tacataactg
     6481 agaatagaga agttcagatc aaggttagga acagagagac agcagaatat gggccaaaca
     6541 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga
     6601 tgcggtcccg ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg
     6661 acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt
     6721 cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact cggcgcgcca
     6781 gtcctccgat agactgcgtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg
     6841 catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg
     6901 tcagcggggg tctttcatgg gtaacagttt cttgaagttg gagaacaaca ttctgagggt
     6961 aggagtcgaa tattaagtaa tcctgactca attagccact gttttgaatc cacatactcc
     7021 aatactcctg aaatagttca ttatggacag cgcagaaaga gctggggaga attgtgaaat
     7081 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     7141 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     7201 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     7261 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     7321 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     7381 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     7441 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     7501 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     7561 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     7621 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     7681 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     7741 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     7801 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     7861 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     7921 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     7981 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     8041 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     8101 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     8161 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     8221 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     8281 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     8341 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     8401 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     8461 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     8521 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     8581 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     8641 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     8701 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     8761 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     8821 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     8881 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     8941 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     9001 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     9061 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     9121 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     9181 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     9241 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     9301 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     9361 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     9421 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     9481 cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac
     9541 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     9601 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     9661 acgacggcgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     9721 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     9781 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     9841 tgccggccac gatgcgtccg gcgtagaggc gattagtcca atttgttaaa gacaggatat
     9901 cagtggtcca ggctctagtt ttgactcaac aatatcacca gctgaagcct atagagtacg
     9961 agccatagat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc
    10021 ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca t
//
