LOCUS       sequence-209253-       11336 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cas9 and sgRNA targeting MTHFD2L.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11336)
  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 11336)
  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: 
            "10.1016/j.molcel.2018.01"; journalName: "Mol Cell"; date: 
            "2018-02-15- 15"; volume: "69"; issue: "4"; pages: "610-621.e5"
COMMENT     Sequence Label: pSECB sgMTHFD2L_1
FEATURES             Location/Qualifiers
     source          1..11336
                     /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    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..2908
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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        2930..3005
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3053..3170
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3269..3480
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3525..3534
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3531..7634
                     /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             3531..7634
                     /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             7635..7682
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7683..7706
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7716..7772
                     /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             7773..8171
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8224..8812
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8277..8297)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8695..8706)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8815..8831)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8816..8832)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9341..9521
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(9583..10171)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9663..9682)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10342..11133,11134..11202))
                     /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     10965..10984
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11203..11307)
                     /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 gagagcatga acggccaggg ttttagagct
     2941 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     3001 ggtgcttttt tgaattctag atcttgagac aaatggcagt attcatccac aattttaaaa
     3061 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     3121 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     3181 acagggacag cagagatcca ctttggcgcc ggcaaaccgc tgttcctagg aatcccgagg
     3241 ccttcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     3301 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     3361 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     3421 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     3481 gtgtcgtgac gcgggacccg acattagcgc tacagcttaa gcgggccacc atggacaaga
     3541 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg
     3601 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     3661 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     3721 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     3781 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     3841 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     3901 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     3961 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     4021 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     4081 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     4141 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     4201 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     4261 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     4321 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     4381 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     4441 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     4501 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     4561 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     4621 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     4681 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     4741 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     4801 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     4861 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     4921 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     4981 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     5041 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     5101 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     5161 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     5221 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     5281 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     5341 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     5401 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     5461 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     5521 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     5581 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     5641 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     5701 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     5761 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     5821 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     5881 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     5941 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     6001 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     6061 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     6121 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     6181 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     6241 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     6301 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     6361 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     6421 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     6481 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     6541 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     6601 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     6661 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     6721 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     6781 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     6841 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     6901 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     6961 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctac
     7021 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     7081 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     7141 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     7201 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     7261 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     7321 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     7381 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     7441 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     7501 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     7561 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     7621 agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct aagaagaaga
     7681 aagattacaa agacgatgac gataagggat ccggcgcaac aaacttctct ctgctgaaac
     7741 aagccggaga tgtcgaagag aatcctggac cgatggccaa gcctttgtct caagaagaat
     7801 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     7861 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     7921 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     7981 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     8041 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
     8101 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     8161 gggagggcta aaaacgttgt tgtttggggt tgaattactc ttctagaccg cggcgcgccc
     8221 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     8281 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     8341 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     8401 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     8461 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     8521 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     8581 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     8641 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     8701 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     8761 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     8821 cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc
     8881 taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt
     8941 cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     9001 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct
     9061 tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg
     9121 gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca
     9181 agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct
     9241 gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca gccgcctagc
     9301 atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct ggttagacca
     9361 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     9421 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     9481 atccctcaga cccttttagt cagtgtggaa aatctctagc agcatgtgag caaaaggcca
     9541 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     9601 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     9661 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     9721 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     9781 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     9841 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     9901 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     9961 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
    10021 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
    10081 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
    10141 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
    10201 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
    10261 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
    10321 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
    10381 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
    10441 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
    10501 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
    10561 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
    10621 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
    10681 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
    10741 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
    10801 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
    10861 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
    10921 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
    10981 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
    11041 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
    11101 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
    11161 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
    11221 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
    11281 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacg
//
