pSECC

LOCUS       pSECC               13907 bp ds-DNA     circular SYN 12-SEP-2022
DEFINITION  3rd generation vector. Expresses a sgRNA of interest, Cas9 and Cre.
ACCESSION   .
VERSION     .
KEYWORDS    pSECC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13907)
  AUTHORS   Sanchez-Rivera FJ, Papagiannakopoulos T, Romero R, Tammela T, Bauer 
            MR, Bhutkar A, Joshi NS, Subbaraj L, Bronson RT, Xue W, Jacks T
  TITLE     Rapid modelling of cooperating genetic events in cancer through 
            somatic genome editing.
  JOURNAL   Nature. 2014 Oct 22. doi: 10.1038/nature13906.
  PUBMED    25337879
REFERENCE   2  (bases 1 to 13907)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 12, 2022 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..13907
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             834..1014
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1061..1186
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1683..1916
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2101..2145
                     /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    2443..2560
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2660..2900
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2660..2680
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2831..2850
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4790..4865
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    4913..5030
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5129..5340
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5385..5394
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             5394..5417
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5424..5444
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5469..9569
                     /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             9570..9617
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9633..9689
                     /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             9693..9713
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9714..10742
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPGIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    10795..11383
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10848..10868)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11266..11277)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11386..11402)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11387..11403)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11912..12092
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(12154..12742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12234..12253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12913..13773)
                     /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     13536..13555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13774..13878)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
      841 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      901 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      961 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     1021 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     1081 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     1141 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     1201 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     1261 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     1321 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     1381 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     1441 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     1501 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat
     1561 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1621 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1681 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1741 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1801 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1861 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1921 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1981 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     2041 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     2101 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     2161 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     2221 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2281 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2341 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2401 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2461 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2521 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2581 cagagatcca gtttggttag taccgggccc gggtcgacta gtgatcagtg tgagggagtg
     2641 taaagctggg tttcccagag agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2701 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
     2761 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2821 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2881 atatatcttg tggaaaggac gaaacaccgg agacggttgt aaatgagcac acaaaataca
     2941 catgctaaaa tattatattc tatgaccttt ataaaatcaa ccaaaatctt ctttttaata
     3001 actttagtat caataattag aatttttatg ttcctttttg caaactttta ataaaaatga
     3061 gcaaaataaa aaaacgctag ttttagtaac tcgcgttgtt ttcttcacct ttaataatag
     3121 ctactccacc acttgttcct aagcggtcag ctcctgcttc aatcattttt tgagcatctt
     3181 caaatgttct aactccacca gctgctttaa ctaaagcatt gtctttaaca actgacttca
     3241 ttagtttaac atcttcaaat gttgcacctg attttgaaaa tcctgttgat gttttaacaa
     3301 attctaatcc agcttcaaca gctatttcac aagctttcat gatttcttct tttgttaata
     3361 aacaattttc cataatacat ttaacaacat gtgatccagc tgcttttttt acagctttca
     3421 tgtcttctaa aactaattca taatttttgt cttttaatgc accaatattt aataccatat
     3481 caatttctgt tgcaccatct ttaattgctt cagaaacttc gaatgctttt gtagctgttg
     3541 tgcatgcacc tagaggaaaa cctacaacat ttgttattcc tacatttgtg ccttttaata
     3601 attctttaca atagcttgtt caatatgaat taacacaaac tgttgcaaaa tcaaattcaa
     3661 ttgcttcatc acataattgt ttaatttcag ctttcgtagc atcttgtttt aataatgtgt
     3721 gatctatata tttgtttagt ttcatttttt ctcctatata ttcattttta attttaattc
     3781 tttaataatt tcgtctactt taactttagc gttttgaaca gattcaccaa cacctataaa
     3841 ataaattttt agtttaggtt cagttccact tgggcgaaca gcaaatcatg acttatcttc
     3901 taaataaaat tttagtaagt cttgtcctgg catattatac attccatcga tgtagtcttc
     3961 aacattaaca actttaagtc cagcaatttg agttaagggt gttgctctca atgatttcat
     4021 taatggttca atttttaatt tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt
     4081 gtaatatgca cccatttctt taaataaatc ttctaaatag tctactaatg ttttattttg
     4141 ttttttataa aatcaagcag cctctgctat taatatagaa gcttgtattc catctttatc
     4201 tctagctgag tcatcaatta catatccata actttcttca taagcaaaaa caaaatttaa
     4261 tccgttatct tcttctttag caatttctct acccattcat ttaaatccag ttaaagtttt
     4321 tacaatatta actccatatt tttcatgagc gattctatca cccaaatcac ttgttacaaa
     4381 acttgaatat agagccggat tttttggaat gctatttaag cgttttagat ttgataattt
     4441 tcaatcaatt aaaattggtc ctgtttgatt tccatctaat cttacaaaat gaccatcatg
     4501 ttttattgcc attccaaatc tgtcagcatc tgggtcattc ataataataa tatctgcatc
     4561 atgtttaata ccatattcaa gcggtatttt tcatgcagga tcaaattctg gatttggatt
     4621 tacaacattt ttaaatgttt catcttcaaa tgcatgctct tcaacctcaa taacgttata
     4681 tcctgattca cgtaatattt ttggggtaaa tttagttcct gttccattaa ctgcgctaaa
     4741 aataattttt aaatcttttt tagcttcttg ctcttttttg tacgtctctg ttttagagct
     4801 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     4861 ggtgcttttt tgaattctag atcttgagac aaatggcagt attcatccac aattttaaaa
     4921 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     4981 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     5041 acagggacag cagagatcca ctttggcgcc ggcaaaccgc tgttcctagg aatcccgagg
     5101 ccttcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     5161 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     5221 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     5281 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     5341 gtgtcgtgac gcgggacccg acattagcgc tacagcttaa gcgggccacc atggattaca
     5401 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
     5461 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     5521 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     5581 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     5641 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     5701 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     5761 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     5821 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     5881 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     5941 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     6001 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     6061 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     6121 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     6181 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     6241 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     6301 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     6361 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     6421 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     6481 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     6541 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     6601 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     6661 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     6721 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     6781 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     6841 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     6901 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     6961 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     7021 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     7081 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     7141 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     7201 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     7261 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     7321 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     7381 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     7441 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     7501 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     7561 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     7621 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     7681 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     7741 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     7801 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     7861 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     7921 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     7981 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     8041 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     8101 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     8161 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     8221 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     8281 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     8341 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     8401 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     8461 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     8521 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     8581 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     8641 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     8701 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     8761 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     8821 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     8881 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     8941 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     9001 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     9061 ctaagtactc cctgttcgag ctggaaaacg gcaggaagag aatgctggcc tctgccggcg
     9121 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     9181 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     9241 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     9301 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     9361 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     9421 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     9481 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     9541 ggatcgacct gtctcagctg ggaggcgaca agcgtcctgc tgctactaag aaagctggtc
     9601 aagctaagaa aaagaaagct agcggcagcg gcgccaccaa cttcagcctg ctgaagcagg
     9661 ccggcgacgt ggaggagaac cccggcccca tgcccaagaa gaagaggaag gtgtccaatt
     9721 tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt gatgaggttc
     9781 gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat acctggaaaa
     9841 tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac cggaaatggt
     9901 ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg cgcggtctgg
     9961 cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt cggtccgggc
    10021 tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc cgaaaagaaa
    10081 acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact gatttcgacc
    10141 aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat ctggcatttc
    10201 tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc agggttaaag
    10261 atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg aaaacgctgg
    10321 ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg gtcgagcgat
    10381 ggatttccgt atctggtgta gctgatgatc cgaataacta cctgttttgc cgggtcagaa
    10441 aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc ctggaaggga
    10501 tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt cagagatacc
    10561 tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc cgcgctggag
    10621 tttcaatacc ggggatcatg caagctggtg gctggaccaa tgtaaatatt gtcatgaact
    10681 atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa gatggcgatt
    10741 agaaacgttg ttgtttgggg ttgaattact cttctagacc gcggcgcgcc cgataatcaa
    10801 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
    10861 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
    10921 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
    10981 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
    11041 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
    11101 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
    11161 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
    11221 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
    11281 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
    11341 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct
    11401 cgatcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc
    11461 tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca
    11521 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
    11581 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg
    11641 gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat
    11701 cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt
    11761 agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat
    11821 ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca
    11881 catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc
    11941 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
    12001 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
    12061 acccttttag tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc
    12121 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
    12181 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
    12241 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
    12301 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
    12361 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
    12421 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
    12481 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
    12541 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
    12601 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
    12661 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
    12721 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
    12781 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
    12841 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
    12901 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
    12961 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
    13021 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
    13081 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
    13141 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
    13201 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
    13261 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
    13321 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
    13381 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
    13441 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
    13501 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
    13561 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
    13621 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
    13681 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
    13741 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
    13801 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
    13861 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacg
//