LOCUS       sequence-243533-       10537 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 10537)
  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 10537)
  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..10537
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    187..528
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             595..1011
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     941..963
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     979..995
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1030..1039
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1039..1104
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1111..1131
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1156..5256
                     /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             1156..5256
                     /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             5257..5304
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5320..5373
                     /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(5386..5388,5389..5391,5392..6105)
                     /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(5692..5711)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6039..6060
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_RNA        complement(6137..6212)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(6290..6309)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     LTR             6538..7052
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    7214..7230
                     /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(7238..7244,7245..7262,7263..7268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7283..7304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7421..7438)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7592..8180)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7672..7691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8351..9142,9143..9211))
                     /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     8974..8993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9212..9316)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9384..9402
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(9440..9462)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     9562..9581
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9775..9797
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9925..9944
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(10144..10537,1..123)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 cgtcgcccgg gtacccgtat tcccaataaa gcctcttgct gtttgcatcc gaatcgtgga
       61 ctcgctgatc cttgggaggg tctcctcaga ttgattgact gcccacctcg ggggtctttc
      121 atttggaggt tccaccgaga tttggagacc cctgcccagg gaccaccgac ccccccgccg
      181 ggaggtaagc tggccagcgg tcgtttcgtg tctgtctctg tctttgtgcg tgtttgtgcc
      241 ggcatctaat gtttgcgcct gcgtctgtac tagttagcta actagctctg tatctggcgg
      301 acccgtggtg gaactgacga gttctgaaca cccggccgca accctgggag acgtcccagg
      361 gactttgggg gccgtttttg tggcccgacc tgaggaaggg agtcgatgtg gaatccgacc
      421 ccgtcaggat atgtggttct ggtaggagac gagaacctaa aacagttccc gcctccgtct
      481 gaatttttgc tttcggtttg gaaccgaagc cgcgcgtctt gtctgctgca gcgctgcagc
      541 atcgttctgt gttgtctctg tctgactgtg tttctgtatt tgtctgaaaa ttagggccag
      601 actgttacca ctcccttaag tttgacctta ggtcactgga aagatgtcga gcggatcgct
      661 cacaaccagt cggtagatgt caagaagaga cgttgggtta ccttctgctc tgcagaatgg
      721 ccaaccttta acgtcggatg gccgcgagac ggcaccttta accgagacct catcacccag
      781 gttaagatca aggtcttttc acctggcccg catggacacc cagaccaggt cccctacatc
      841 gtgacctggg aagccttggc ttttgacccc cctccctggg tcaagccctt tgtacaccct
      901 aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc cccttgaacc tcctcgttcg
      961 accccgcctc gatcctccct ttatccagcc ctcactcctt ctctaggcgc cggaattaga
     1021 tctctcgagg ccaccatgga ctataaggac cacgacggag actacaagga tcatgatatt
     1081 gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac
     1141 ggagtcccag cagccgacaa gaagtacagc atcggcctgg acatcggcac caactctgtg
     1201 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
     1261 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
     1321 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
     1381 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
     1441 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
     1501 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
     1561 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
     1621 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
     1681 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
     1741 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
     1801 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
     1861 ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
     1921 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
     1981 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
     2041 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
     2101 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
     2161 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
     2221 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
     2281 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
     2341 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
     2401 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
     2461 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
     2521 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
     2581 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
     2641 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
     2701 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
     2761 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
     2821 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
     2881 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
     2941 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
     3001 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
     3061 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
     3121 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
     3181 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
     3241 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
     3301 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
     3361 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
     3421 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
     3481 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
     3541 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
     3601 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
     3661 gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg
     3721 ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg
     3781 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
     3841 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
     3901 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
     3961 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
     4021 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
     4081 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
     4141 ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac
     4201 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
     4261 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
     4321 atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
     4381 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
     4441 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
     4501 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
     4561 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
     4621 ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag
     4681 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
     4741 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
     4801 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
     4861 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
     4921 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
     4981 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
     5041 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
     5101 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
     5161 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
     5221 gagacacgga tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag
     5281 gccggccagg caaaaaagaa aaaggaattc ggatccggag agggcagagg aagtcttcta
     5341 acatgcggtg acgtggagga gaatcccggc cctatggcca caaccatggt gagcaagggc
     5401 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     5461 cacaagttca gcgtgtctgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     5521 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     5581 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     5641 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     5701 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     5761 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     5821 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggcgaac
     5881 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     5941 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     6001 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     6061 accgccgccg ggatcactct cggcatggac gagctgtaca agtaatgaat taattaagaa
     6121 ttgcggccgc aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc
     6181 ttattttaac ttgctatttc tagctctaaa acaggtcttc tcgaagaccc cggtgtttcg
     6241 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
     6301 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
     6361 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat
     6421 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa agtcgacctg
     6481 cagccaagct tatcgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
     6541 aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg
     6601 aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag agacagcaga
     6661 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
     6721 agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc
     6781 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc
     6841 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct
     6901 cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtgt atccaataaa
     6961 ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag
     7021 tgattgacta cccgtcagcg ggggtctttc atgggtaaca gtttcttgaa gttggagaac
     7081 aacattctga gggtaggagt cgaatattaa gtaatcctga ctcaattagc cactgttttg
     7141 aatccacata ctccaatact cctgaaatag ttcattatgg acagcgcaga aagagctggg
     7201 gagaattgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     7261 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     7321 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     7381 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     7441 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     7501 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     7561 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     7621 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     7681 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     7741 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     7801 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     7861 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     7921 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     7981 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     8041 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     8101 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     8161 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     8221 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     8281 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     8341 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     8401 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     8461 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     8521 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     8581 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     8641 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     8701 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     8761 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     8821 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     8881 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     8941 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     9001 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     9061 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     9121 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     9181 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     9241 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     9301 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     9361 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     9421 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     9481 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     9541 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     9601 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat
     9661 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     9721 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     9781 cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     9841 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     9901 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     9961 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggcgattag tccaatttgt
    10021 taaagacagg atatcagtgg tccaggctct agttttgact caacaatatc accagctgaa
    10081 gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc agaaaaaggg
    10141 gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa
    10201 ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg aacagagaga
    10261 cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
    10321 caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag aaccatcaga
    10381 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
    10441 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
    10501 aacccctcac tcggcgcgcc agtcctccga tagactg
//
