LOCUS       sequence-201284-       13190 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express spCas9 with sgRNA, blast resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13190)
  AUTHORS   Minton DR, Nam M, McLaughlin DJ, Shin J, Bayraktar EC, Alvarez SW, 
            Sviderskiy VO, Papagiannakopoulos T, Sabatini DM, Birsoy K, 
            Possemato R
  TITLE     Serine Catabolism by SHMT2 Is Required for Proper Mitochondrial 
            Translation Initiation and Maintenance of Formylmethionyl-tRNAs.
  JOURNAL   Mol Cell. 2018 Feb 15;69(4):610-621.e5. doi: 
            10.1016/j.molcel.2018.01.024.
  PUBMED    29452640
REFERENCE   2  (bases 1 to 13190)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2018.01"; journalName: "Mol Cell"; date: 
            "2018-02-15- 15"; volume: "69"; issue: "4"; pages: "610-621.e5"
COMMENT     Sequence Label: pSECB
FEATURES             Location/Qualifiers
     source          1..13190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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    1685..1918
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2103..2147
                     /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    2445..2562
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2655..2903
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2655..2675
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2826..2845
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4785..4860
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    4908..5025
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5124..5335
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5380..5389
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5386..9489
                     /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             5386..9489
                     /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             9490..9537
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9538..9561
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             9571..9627
                     /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             9628..10026
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    10079..10667
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10132..10152)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10550..10561)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10670..10686)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10671..10687)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11196..11376
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(11438..12026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11518..11537)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12197..12988,12989..13057))
                     /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     12820..12839
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13058..13162)
                     /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 ccgcgctgat cttcagacct ggaggaggag
     1561 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     1621 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     1681 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     1741 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     1801 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     1861 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     1921 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     1981 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     2041 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     2101 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     2161 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     2221 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     2281 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     2341 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac
     2401 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     2461 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     2521 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     2581 agcagagatc cagtttggtt agtaccgggc ccgggtcgac tagtgatcag tgtgagggag
     2641 tgtaaagctg gtttgagggc ctatttccca tgattccttc atatttgcat atacgataca
     2701 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
     2761 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     2821 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     2881 tcttgtggaa aggacgaaac accggagacg gttgtaaatg agcacacaaa atacacatgc
     2941 taaaatatta tattctatga cctttataaa atcaaccaaa atcttctttt taataacttt
     3001 agtatcaata attagaattt ttatgttcct ttttgcaaac ttttaataaa aatgagcaaa
     3061 ataaaaaaac gctagtttta gtaactcgcg ttgttttctt cacctttaat aatagctact
     3121 ccaccacttg ttcctaagcg gtcagctcct gcttcaatca ttttttgagc atcttcaaat
     3181 gttctaactc caccagctgc tttaactaaa gcattgtctt taacaactga cttcattagt
     3241 ttaacatctt caaatgttgc acctgatttt gaaaatcctg ttgatgtttt aacaaattct
     3301 aatccagctt caacagctat ttcacaagct ttcatgattt cttcttttgt taataaacaa
     3361 ttttccataa tacatttaac aacatgtgat ccagctgctt tttttacagc tttcatgtct
     3421 tctaaaacta attcataatt tttgtctttt aatgcaccaa tatttaatac catatcaatt
     3481 tctgttgcac catctttaat tgcttcagaa acttcgaatg cttttgtagc tgttgtgcat
     3541 gcacctagag gaaaacctac aacatttgtt attcctacat ttgtgccttt taataattct
     3601 ttacaatagc ttgttcaata tgaattaaca caaactgttg caaaatcaaa ttcaattgct
     3661 tcatcacata attgtttaat ttcagctttc gtagcatctt gttttaataa tgtgtgatct
     3721 atatatttgt ttagtttcat tttttctcct atatattcat ttttaatttt aattctttaa
     3781 taatttcgtc tactttaact ttagcgtttt gaacagattc accaacacct ataaaataaa
     3841 tttttagttt aggttcagtt ccacttgggc gaacagcaaa tcatgactta tcttctaaat
     3901 aaaattttag taagtcttgt cctggcatat tatacattcc atcgatgtag tcttcaacat
     3961 taacaacttt aagtccagca atttgagtta agggtgttgc tctcaatgat ttcattaatg
     4021 gttcaatttt taatttcttt tcttctggtt taaaattcaa gtttaaagtg aaagtgtaat
     4081 atgcacccat ttctttaaat aaatcttcta aatagtctac taatgtttta ttttgttttt
     4141 tataaaatca agcagcctct gctattaata tagaagcttg tattccatct ttatctctag
     4201 ctgagtcatc aattacatat ccataacttt cttcataagc aaaaacaaaa tttaatccgt
     4261 tatcttcttc tttagcaatt tctctaccca ttcatttaaa tccagttaaa gtttttacaa
     4321 tattaactcc atatttttca tgagcgattc tatcacccaa atcacttgtt acaaaacttg
     4381 aatatagagc cggatttttt ggaatgctat ttaagcgttt tagatttgat aattttcaat
     4441 caattaaaat tggtcctgtt tgatttccat ctaatcttac aaaatgacca tcatgtttta
     4501 ttgccattcc aaatctgtca gcatctgggt cattcataat aataatatct gcatcatgtt
     4561 taataccata ttcaagcggt atttttcatg caggatcaaa ttctggattt ggatttacaa
     4621 catttttaaa tgtttcatct tcaaatgcat gctcttcaac ctcaataacg ttatatcctg
     4681 attcacgtaa tatttttggg gtaaatttag ttcctgttcc attaactgcg ctaaaaataa
     4741 tttttaaatc ttttttagct tcttgctctt ttttgtacgt ctctgtttta gagctagaaa
     4801 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     4861 ttttttgaat tctagatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa
     4921 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     4981 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     5041 gacagcagag atccactttg gcgccggcaa accgctgttc ctaggaatcc cgaggccttc
     5101 gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     5161 ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga
     5221 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     5281 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc
     5341 gtgacgcggg acccgacatt agcgctacag cttaagcggg ccaccatgga caagaagtac
     5401 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     5461 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     5521 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     5581 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     5641 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     5701 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     5761 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     5821 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     5881 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     5941 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     6001 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     6061 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     6121 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     6181 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     6241 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     6301 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     6361 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     6421 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     6481 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     6541 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     6601 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     6661 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     6721 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     6781 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     6841 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     6901 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     6961 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     7021 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     7081 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     7141 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     7201 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     7261 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     7321 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     7381 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     7441 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     7501 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     7561 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     7621 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     7681 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     7741 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     7801 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     7861 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     7921 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     7981 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     8041 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     8101 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     8161 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     8221 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     8281 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     8341 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     8401 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     8461 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     8521 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     8581 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     8641 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     8701 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     8761 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     8821 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctactgggg
     8881 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     8941 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     9001 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     9061 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     9121 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     9181 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     9241 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     9301 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     9361 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     9421 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
     9481 ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa gaagaaagat
     9541 tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct gaaacaagcc
     9601 ggagatgtcg aagagaatcc tggaccgatg gccaagcctt tgtctcaaga agaatccacc
     9661 ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc
     9721 gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt
     9781 actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc
     9841 aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga
     9901 cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt
     9961 gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag
    10021 ggctaaaaac gttgttgttt ggggttgaat tactcttcta gaccgcggcg cgcccgataa
    10081 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
    10141 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
    10201 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
    10261 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
    10321 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
    10381 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
    10441 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
    10501 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
    10561 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
    10621 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg
    10681 acctcgatcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca
    10741 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac
    10801 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
    10861 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc
    10921 tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag
    10981 ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga
    11041 aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg
    11101 ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc
    11161 atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct
    11221 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
    11281 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
    11341 tcagaccctt ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa
    11401 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
    11461 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
    11521 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
    11581 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
    11641 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
    11701 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
    11761 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
    11821 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
    11881 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
    11941 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
    12001 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
    12061 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
    12121 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
    12181 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
    12241 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
    12301 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
    12361 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
    12421 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
    12481 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
    12541 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    12601 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    12661 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    12721 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    12781 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    12841 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    12901 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    12961 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
    13021 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
    13081 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
    13141 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
//
