LOCUS       sequence-209255-       11590 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express Cas9 and sgRNA targeting MTHFD2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11590)
  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 11590)
  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"; 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: pLenti CRISPR sgMTHFD2_1
FEATURES             Location/Qualifiers
     source          1..11590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    75..96
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(111..116,117..134,135..141)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    149..165
                     /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)."
     primer_bind     154..176
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     173..189
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     173..189
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     208..228
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        210..228
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        256..482
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             483..663
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    710..835
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1328..1561
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1746..1790
                     /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"
     promoter        1968..2216
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1968..1988
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2139..2158
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2238..2313
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2361..2478
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2547..2758
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2778..2787
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2787..2810
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2817..2837
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2862..6962
                     /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             2862..6962
                     /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             6963..7010
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7026..7082
                     /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             7083..7682
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7083..7102)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7579..7599
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7698..8286
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7751..7771)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8169..8180)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8358..8591
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8669..8790
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8706..8725)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8760..8779
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8830..8965
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8985..9004)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8986..9004)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9014..9031)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9014..9030)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9023..9045)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9172..9627
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9259..9278)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9469..9490
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9653..9757
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9758..9826,9827..10618)
                     /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     complement(9976..9995)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10789..11377
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11278..11297
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11531..11548
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
       61 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
      121 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      181 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
      241 gagctgcaag cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga
      301 tgagttagca acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag
      361 taaggtggta cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac
      421 gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa
      481 acgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
      541 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
      601 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
      661 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg
      721 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac
      781 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat
      841 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa
      901 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
      961 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc
     1021 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
     1081 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga
     1141 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag
     1201 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac
     1261 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag
     1321 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag
     1381 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga
     1441 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca
     1501 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc
     1561 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta
     1621 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca
     1681 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc
     1741 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt
     1801 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg
     1861 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt
     1921 caccattatc gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc
     1981 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta
     2041 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     2101 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     2161 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcga
     2221 agggagcagc tgtgcgcgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     2281 tatcaacttg aaaaagtggc accgagtcgg tgcttttttg aattctagat cttgagacaa
     2341 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     2401 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     2461 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg
     2521 ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     2581 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg
     2641 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     2701 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt
     2761 gtcgtgacgc gggatccgcc accatggatt acaaagacga tgacgataag atggccccaa
     2821 agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg
     2881 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     2941 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     3001 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     3061 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     3121 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     3181 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     3241 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     3301 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     3361 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     3421 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     3481 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     3541 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     3601 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     3661 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     3721 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     3781 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     3841 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     3901 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     3961 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     4021 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     4081 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     4141 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     4201 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     4261 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     4321 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     4381 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     4441 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     4501 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     4561 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     4621 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     4681 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     4741 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     4801 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     4861 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     4921 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     4981 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     5041 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     5101 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     5161 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     5221 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     5281 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     5341 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     5401 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     5461 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     5521 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     5581 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     5641 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     5701 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     5761 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     5821 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     5881 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     5941 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     6001 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     6061 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     6121 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     6181 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     6241 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     6301 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     6361 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     6421 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     6481 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     6541 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     6601 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     6661 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     6721 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     6781 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     6841 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     6901 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     6961 acaagcgtcc tgctgctact aagaaagctg gtcaagctaa gaaaaagaaa gctagcggca
     7021 gcggcgccac caacttcagc ctgctgaagc aggccggcga cgtggaggag aaccccggcc
     7081 ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     7141 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     7201 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     7261 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     7321 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     7381 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     7441 agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg
     7501 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     7561 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     7621 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     7681 gaacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     7741 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     7801 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     7861 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     7921 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     7981 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     8041 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     8101 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     8161 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     8221 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     8281 ccccgcgtcg actttaagac caatgactta caaggcagct gtagatctta gccacttttt
     8341 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt
     8401 tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     8461 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     8521 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     8581 aatctctagc agtacgtata gtagttcatg tcatcttatt attcagtatt tataacttgc
     8641 aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa
     8701 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     8761 tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta
     8821 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     8881 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     8941 tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt
     9001 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     9061 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     9121 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct
     9181 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     9241 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     9301 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     9361 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     9421 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     9481 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     9541 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     9601 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     9661 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     9721 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     9781 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     9841 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     9901 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     9961 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
    10021 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
    10081 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
    10141 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
    10201 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
    10261 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
    10321 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
    10381 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
    10441 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
    10501 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
    10561 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
    10621 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
    10681 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
    10741 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
    10801 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
    10861 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
    10921 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
    10981 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
    11041 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
    11101 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
    11161 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
    11221 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
    11281 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
    11341 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
    11401 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
    11461 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
    11521 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
    11581 tctccccgcg
//
