LOCUS       sequence-209275-       11590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cas9 and sgRNA targeting SHMT1.
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 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 sgSHMT1_2
FEATURES             Location/Qualifiers
     source          1..11590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1058..1284
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1285..1465
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1512..1637
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2130..2363
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2548..2592
                     /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        2770..3018
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2770..2790
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2941..2960
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3040..3115
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3163..3280
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3349..3560
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3580..3589
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3589..3612
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3619..3639
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3664..7764
                     /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             3664..7764
                     /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             7765..7812
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7828..7884
                     /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             7885..8484
                     /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(7885..7904)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8381..8401
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8500..9088
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8553..8573)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8971..8982)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9160..9393
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    9471..9592
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9508..9527)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9562..9581
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      9632..9767
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9787..9806)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9788..9806)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9816..9833)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9816..9832)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9825..9847)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9974..10429
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10061..10080)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10271..10292
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10455..10559
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10560..10628,10629..11420)
                     /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(10778..10797)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
     1081 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
     1141 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
     1201 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
     1261 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
     1321 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1381 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1441 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1501 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1561 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1621 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1681 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1741 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1801 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1861 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1921 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1981 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     2041 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     2101 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     2161 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     2221 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     2281 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     2341 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     2401 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     2461 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     2521 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     2581 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     2641 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     2701 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     2761 ggacccagag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2821 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2881 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2941 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     3001 tggaaaggac gaaacaccgg aaggagagta accggcagag ttttagagct agaaatagca
     3061 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     3121 tgaattctag atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     3181 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     3241 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     3301 cagagatcca ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca
     3361 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
     3421 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     3481 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     3541 ggtttgccgc cagaacacag gtgtcgtgac gcgggatccg ccaccatgga ttacaaagac
     3601 gatgacgata agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca
     3661 gccgacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
     3721 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     3781 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     3841 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     3901 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     3961 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     4021 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     4081 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     4141 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     4201 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     4261 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     4321 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
     4381 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     4441 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     4501 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     4561 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     4621 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     4681 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     4741 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     4801 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     4861 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     4921 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     4981 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     5041 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     5101 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     5161 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     5221 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     5281 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     5341 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     5401 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     5461 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     5521 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     5581 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     5641 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     5701 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     5761 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     5821 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     5881 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     5941 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     6001 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     6061 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     6121 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     6181 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     6241 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     6301 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     6361 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     6421 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     6481 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     6541 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     6601 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     6661 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     6721 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     6781 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     6841 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     6901 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     6961 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     7021 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     7081 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     7141 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     7201 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     7261 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     7321 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     7381 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     7441 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     7501 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     7561 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     7621 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     7681 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     7741 gacctgtctc agctgggagg cgacaagcgt cctgctgcta ctaagaaagc tggtcaagct
     7801 aagaaaaaga aagctagcgg cagcggcgcc accaacttca gcctgctgaa gcaggccggc
     7861 gacgtggagg agaaccccgg ccccatgacc gagtacaagc ccacggtgcg cctcgccacc
     7921 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     7981 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     8041 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     8101 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     8161 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     8221 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc
     8281 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     8341 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     8401 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     8461 atgacccgca agcccggtgc ctgaacgcgt taagtcgaca atcaacctct ggattacaaa
     8521 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     8581 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     8641 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     8701 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     8761 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     8821 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     8881 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     8941 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     9001 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     9061 cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag
     9121 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     9181 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     9241 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     9301 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     9361 gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta
     9421 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     9481 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     9541 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     9601 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     9661 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     9721 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
     9781 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     9841 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     9901 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     9961 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
    10021 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
    10081 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
    10141 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
    10201 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
    10261 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
    10321 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
    10381 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
    10441 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
    10501 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
    10561 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
    10621 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
    10681 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
    10741 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
    10801 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
    10861 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
    10921 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
    10981 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
    11041 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
    11101 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
    11161 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
    11221 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
    11281 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
    11341 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
    11401 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
    11461 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
    11521 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
    11581 aaggatcttc
//
