LOCUS       sequence-318493-       13489 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Lentiviral construct for the expression of SpCas9 driven by EFS 
            promoter in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13489)
  AUTHORS   Park J, Lim JM, Jung I, Heo SJ, Park J, Chang Y, Kim HK, Jung D, Yu 
            JH, Min S, Yoon S, Cho SR, Park T, Kim HH
  TITLE     Recording of elapsed time and temporal information about biological 
            events using Cas9.
  JOURNAL   Cell. 2021 Jan 28. pii: S0092-8674(21)00014-3. doi: 
            10.1016/j.cell.2021.01.014.
  PUBMED    33539780
REFERENCE   2  (bases 1 to 13489)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2021 
            Jan 28. pii: S0092-8674(21)00014-3. doi: 
            10.1016/j.cell.2021.01.014."
COMMENT     Sequence Label: Lenti_EFS-Cas9-P2A-HygR
FEATURES             Location/Qualifiers
     source          1..13489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        119..498
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        500..698
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     653..673
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             716..896
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    943..1068
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1561..1794
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1979..2023
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2321..2438
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2531..2742
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2761..2770
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2767..6870
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2767..6870
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6871..6918
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6919..6942
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6952..7008
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7009..8037
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MAKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     misc_feature    8062..8650
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8115..8135)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8533..8544)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8653..8669)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8654..8670)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9175..9355
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(9381..9398)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9387..9611
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9657..10085
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9744..9763)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9954..9975
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10094..10114)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10099..10428
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10279..10414
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10341..10360
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10476..10523
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10542..10916
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    11046..11167
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11083..11102)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11137..11156
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11216..11232)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11216..11232)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11229..11251)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11240..11256
                     /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(11264..11270,11271..11288,11289..11294))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11309..11330
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11447..11464)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11618..12206)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11698..11717)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12377..13168,13169..13237))
                     /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     13000..13019
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13238..13342)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(13417..13436)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
       61 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      121 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      181 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      241 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      301 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      361 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      421 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      481 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      541 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      601 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      661 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
      721 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      781 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      841 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      901 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      961 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     1021 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     1081 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     1141 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     1201 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1261 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1321 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1381 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     1441 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1501 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1561 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1621 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1681 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1741 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     1801 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     1861 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     1921 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     1981 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     2041 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     2101 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     2161 atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga
     2221 agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg
     2281 tgcgccaatt ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     2341 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     2401 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     2461 gagatccagt ttggttaatt agctagctag gtcttgaaag gagtgggaat tggctccggt
     2521 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     2581 ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     2641 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     2701 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagagcgct
     2761 gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     2821 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     2881 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     2941 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     3001 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     3061 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     3121 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     3181 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     3241 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     3301 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     3361 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     3421 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     3481 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     3541 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     3601 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     3661 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     3721 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     3781 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     3841 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     3901 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     3961 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     4021 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     4081 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     4141 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     4201 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     4261 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     4321 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     4381 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     4441 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     4501 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     4561 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     4621 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     4681 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     4741 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     4801 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     4861 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     4921 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     4981 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     5041 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     5101 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     5161 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     5221 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     5281 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     5341 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     5401 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     5461 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     5521 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     5581 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     5641 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     5701 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     5761 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     5821 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     5881 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     5941 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     6001 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     6061 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     6121 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     6181 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     6241 agtgtgaaag agctactggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     6301 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     6361 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     6421 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     6481 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     6541 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     6601 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     6661 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     6721 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     6781 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     6841 cggatcgacc tgtctcagct gggaggcgac aagcgacctg ccgccacaaa gaaggctgga
     6901 caggctaaga agaagaaaga ttacaaagac gatgacgata agggatccgg cgcaacaaac
     6961 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgat ggccaaaaag
     7021 cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtgtcc
     7081 gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
     7141 cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt
     7201 tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggagttc
     7261 agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg
     7321 cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggcaatgga tgcgatcgct
     7381 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
     7441 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa
     7501 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt
     7561 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat
     7621 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg
     7681 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
     7741 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg
     7801 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc
     7861 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact
     7921 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa
     7981 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatgagaa
     8041 ttcgatatca agcttatcgg taatcaacct ctggattaca aaatttgtga aagattgact
     8101 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     8161 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     8221 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     8281 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     8341 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     8401 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     8461 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     8521 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     8581 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     8641 gcctccccgc atcgataccg tcgacctcga gacctagaaa aacatggagc aatcacaagt
     8701 agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga ggaggaggag
     8761 gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa ggcagctgta
     8821 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     8881 agacaagata tccttgatct gtggatctac cacacacaag gctacttccc tgattggcag
     8941 aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg ctacaagcta
     9001 gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac ccgcttgtta
     9061 caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga gtggaggttt
     9121 gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg tactgggtct
     9181 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     9241 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     9301 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc
     9361 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     9421 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     9481 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     9541 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     9601 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     9661 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     9721 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     9781 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     9841 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     9901 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     9961 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
    10021 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
    10081 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
    10141 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
    10201 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
    10261 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
    10321 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
    10381 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
    10441 gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt
    10501 atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg
    10561 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
    10621 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
    10681 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
    10741 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
    10801 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
    10861 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
    10921 tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
    10981 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
    11041 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
    11101 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
    11161 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
    11221 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
    11281 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
    11341 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
    11401 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
    11461 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
    11521 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
    11581 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
    11641 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
    11701 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
    11761 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
    11821 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
    11881 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
    11941 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
    12001 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
    12061 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
    12121 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
    12181 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
    12241 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
    12301 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
    12361 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
    12421 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
    12481 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
    12541 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
    12601 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
    12661 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
    12721 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    12781 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    12841 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    12901 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    12961 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    13021 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    13081 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    13141 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
    13201 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
    13261 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
    13321 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
    13381 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
    13441 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtag
//
