LOCUS       sequence-209331-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cas9 and sgRNA targeting MTHFD1L.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  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 13013)
  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: pLenti CRISPR V2 sgMTHFD1L_2
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /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    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2371..2619
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2371..2391
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2542..2561
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2641..2716
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2778..2989
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3008..3017
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3014..7117
                     /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             3014..7117
                     /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             7118..7165
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7166..7189
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7199..7255
                     /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             7256..7852
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7749..7769
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7868..8456
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7921..7941)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8339..8350)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8528..8761
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8787..8804)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8793..9017
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9063..9491
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9150..9169)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9360..9381
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9500..9520)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9505..9834
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9685..9820
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9747..9766
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9882..9929
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9948..10322
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    10452..10573
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10489..10508)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10543..10562
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10622..10638)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10622..10638)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10635..10657)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10646..10662
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(10670..10676,10677..10694,10695..10700))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10715..10736
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10853..10870)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11024..11612)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11104..11123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11783..12574,12575..12643))
                     /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     12406..12425
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12644..12748)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12823..12842)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2341 cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat tccttcatat
     2401 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
     2461 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     2521 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     2581 ttcttggctt tatatatctt gtggaaagga cgaaacaccg gggagggcct gcttggacag
     2641 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     2701 ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg
     2761 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     2821 aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     2881 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     2941 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggttcta
     3001 gagcgctgcc accatggaca agaagtacag catcggcctg gacatcggca ccaactctgt
     3061 gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg
     3121 caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg
     3181 cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
     3241 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
     3301 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
     3361 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
     3421 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
     3481 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
     3541 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
     3601 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
     3661 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
     3721 cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
     3781 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
     3841 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
     3901 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
     3961 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
     4021 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
     4081 gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat
     4141 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
     4201 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
     4261 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
     4321 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
     4381 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
     4441 gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac
     4501 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
     4561 gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
     4621 gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa
     4681 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
     4741 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
     4801 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
     4861 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
     4921 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
     4981 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
     5041 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
     5101 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
     5161 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
     5221 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
     5281 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
     5341 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
     5401 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
     5461 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
     5521 cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt
     5581 gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
     5641 gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa
     5701 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
     5761 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
     5821 ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat
     5881 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
     5941 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
     6001 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
     6061 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
     6121 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
     6181 gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa
     6241 gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa
     6301 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
     6361 cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
     6421 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
     6481 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
     6541 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
     6601 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
     6661 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
     6721 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
     6781 gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt
     6841 ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa
     6901 gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
     6961 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
     7021 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
     7081 cgagacacgg atcgacctgt ctcagctggg aggcgacaag cgacctgccg ccacaaagaa
     7141 ggctggacag gctaagaaga agaaagatta caaagacgat gacgataagg gatccggcgc
     7201 aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccgaccga
     7261 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     7321 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     7381 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     7441 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     7501 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     7561 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     7621 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     7681 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     7741 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     7801 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta
     7861 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     7921 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     7981 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     8041 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     8101 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     8161 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     8221 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     8281 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     8341 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     8401 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
     8461 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     8521 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     8581 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     8641 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     8701 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     8761 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     8821 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     8881 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     8941 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     9001 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     9061 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     9121 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     9181 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     9241 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     9301 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     9361 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     9421 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     9481 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     9541 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     9601 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     9661 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     9721 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     9781 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     9841 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg
     9901 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga
     9961 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
    10021 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
    10081 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
    10141 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
    10201 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
    10261 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
    10321 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
    10381 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
    10441 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
    10501 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
    10561 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
    10621 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
    10681 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
    10741 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
    10801 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
    10861 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
    10921 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
    10981 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
    11041 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
    11101 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
    11161 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
    11221 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
    11281 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
    11341 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
    11401 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
    11461 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
    11521 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
    11581 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
    11641 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
    11701 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
    11761 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
    11821 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
    11881 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
    11941 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
    12001 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
    12061 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
    12121 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
    12181 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
    12241 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
    12301 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
    12361 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
    12421 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
    12481 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
    12541 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
    12601 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
    12661 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
    12721 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
    12781 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
    12841 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
    12901 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
    12961 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
