LOCUS       sequence-238580-       10538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains spCas9-2A-GFP and guide RNA scaffold in single retroviral 
            vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10538)
  AUTHORS   Li H, Shi J, Huang NJ, Pishesha N, Natarajan A, Eng JC, Lodish HF
  TITLE     Efficient CRISPR-Cas9 mediated gene disruption in primary erythroid 
            progenitor cells.
  JOURNAL   Haematologica. 2016 Jun;101(6):e216-9. doi: 
            10.3324/haematol.2015.135723. Epub 2016 Mar 11.
  PUBMED    26969085
REFERENCE   2  (bases 1 to 10538)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.3324/haematol.2015.135723"; journalName: "Haematologica"; date: 
            "2016-06"; volume: "101"; issue: "6"; pages: "e216-9"
COMMENT     Sequence Label: pMSCV-Cas9-2A-GFP-sgRNA
FEATURES             Location/Qualifiers
     source          1..10538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             43..559
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    623..964
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1031..1447
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1377..1399
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1415..1431
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1466..1475
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1475..1540
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1547..1567
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1592..5692
                     /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             1592..5692
                     /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             5693..5740
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5756..5809
                     /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             join(5822..5824,5825..5827,5828..6541)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6128..6147)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6475..6496
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_RNA        complement(6573..6648)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(6726..6745)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     LTR             6975..7489
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    7651..7667
                     /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(7675..7681,7682..7699,7700..7705))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7720..7741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7858..7875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8029..8617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8109..8128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8788..9579,9580..9648))
                     /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     9411..9430
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9649..9753)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9821..9839
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(9877..9899)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     9999..10018
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     10212..10234
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     10362..10381
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag accccacctg
       61 taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa tacataactg
      121 agaatagaga agttcagatc aaggttagga acagagagac agcagaatat gggccaaaca
      181 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga
      241 tgcggtcccg ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg
      301 acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt
      361 cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact cggcgcgcca
      421 gtcctccgat agactgcgtc gcccgggtac ccgtattccc aataaagcct cttgctgttt
      481 gcatccgaat cgtggactcg ctgatccttg ggagggtctc ctcagattga ttgactgccc
      541 acctcggggg tctttcattt ggaggttcca ccgagatttg gagacccctg cccagggacc
      601 accgaccccc ccgccgggag gtaagctggc cagcggtcgt ttcgtgtctg tctctgtctt
      661 tgtgcgtgtt tgtgccggca tctaatgttt gcgcctgcgt ctgtactagt tagctaacta
      721 gctctgtatc tggcggaccc gtggtggaac tgacgagttc tgaacacccg gccgcaaccc
      781 tgggagacgt cccagggact ttgggggccg tttttgtggc ccgacctgag gaagggagtc
      841 gatgtggaat ccgaccccgt caggatatgt ggttctggta ggagacgaga acctaaaaca
      901 gttcccgcct ccgtctgaat ttttgctttc ggtttggaac cgaagccgcg cgtcttgtct
      961 gctgcagcgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc
     1021 tgaaaattag ggccagactg ttaccactcc cttaagtttg accttaggtc actggaaaga
     1081 tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt gggttacctt
     1141 ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca cctttaaccg
     1201 agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg gacacccaga
     1261 ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc cctgggtcaa
     1321 gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt ctctccccct
     1381 tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca ctccttctct
     1441 aggcgccgga attagatctc tcgaggccac catggactat aaggaccacg acggagacta
     1501 caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg
     1561 gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat
     1621 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     1681 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     1741 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     1801 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     1861 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     1921 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     1981 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     2041 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     2101 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     2161 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     2221 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     2281 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     2341 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     2401 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     2461 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     2521 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     2581 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     2641 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     2701 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     2761 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     2821 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     2881 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     2941 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     3001 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     3061 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     3121 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     3181 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     3241 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     3301 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     3361 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     3421 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     3481 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     3541 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     3601 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     3661 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     3721 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     3781 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     3841 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     3901 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     3961 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     4021 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     4081 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     4141 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     4201 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     4261 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     4321 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     4381 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     4441 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     4501 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     4561 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     4621 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     4681 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     4741 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     4801 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     4861 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     4921 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     4981 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     5041 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     5101 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     5161 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     5221 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     5281 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     5341 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     5401 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     5461 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     5521 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     5581 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     5641 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc
     5701 ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag gaattcggat ccggagaggg
     5761 cagaggaagt cttctaacat gcggtgacgt ggaggagaat cccggcccta tggccacaac
     5821 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     5881 cggcgacgta aacggccaca agttcagcgt gtctggcgag ggcgagggcg atgccaccta
     5941 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     6001 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     6061 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     6121 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     6181 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     6241 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     6301 cggcatcaag gcgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     6361 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     6421 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     6481 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     6541 atgaattaat taagaattgc ggccgcaaaa aagcaccgac tcggtgccac tttttcaagt
     6601 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacag gtcttctcga
     6661 agaccccggt gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc
     6721 aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc
     6781 caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca
     6841 aattaattcc aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca
     6901 tgggaaagtc gaccctgcag ccaagcttat cgataaaata aaagatttta tttagtctcc
     6961 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
     7021 cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg
     7081 aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
     7141 ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag
     7201 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
     7261 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
     7321 aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta
     7381 cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg
     7441 ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcatg ggtaacagtt
     7501 tcttgaagtt ggagaacaac attctgaggg taggagtcga atattaagta atcctgactc
     7561 aattagccac tgttttgaat ccacatactc caatactcct gaaatagttc attatggaca
     7621 gcgcagaaag agctggggag aattgtgaaa ttgttatccg ctcacaattc cacacaacat
     7681 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     7741 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7801 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     7861 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     7921 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     7981 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     8041 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     8101 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     8161 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     8221 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     8281 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     8341 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     8401 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     8461 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     8521 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     8581 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     8641 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     8701 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     8761 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     8821 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     8881 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     8941 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     9001 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     9061 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     9121 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     9181 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     9241 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     9301 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     9361 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     9421 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     9481 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     9541 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     9601 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     9661 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     9721 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     9781 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     9841 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     9901 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     9961 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
    10021 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
    10081 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
    10141 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
    10201 gccagggttt tcccagtcac gacgttgtaa aacgacggcg caaggaatgg tgcatgcaag
    10261 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
    10321 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
    10381 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
    10441 cgattagtcc aatttgttaa agacaggata tcagtggtcc aggctctagt tttgactcaa
    10501 caatatcacc agctgaagcc tatagagtac gagccata
//
