LOCUS       sequence-230320-       11590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cas9 and sgRNA targeting SHMT2.
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 sgSHMT2_1
FEATURES             Location/Qualifiers
     source          1..11590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     98..115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    232..253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(268..273,274..291,292..298)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    306..322
                     /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     311..333
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     330..346
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     330..346
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     365..385
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        367..385
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        413..639
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             640..820
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    867..992
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1485..1718
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1903..1947
                     /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        2125..2373
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2125..2145
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2296..2315
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2395..2470
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2518..2635
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2704..2915
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2935..2944
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2944..2967
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2974..2994
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3019..7119
                     /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             3019..7119
                     /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             7120..7167
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7183..7239
                     /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             7240..7839
                     /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(7240..7259)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7736..7756
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7855..8443
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7908..7928)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8326..8337)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8515..8748
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8826..8947
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8863..8882)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8917..8936
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8987..9122
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9142..9161)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9143..9161)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9171..9188)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9171..9187)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9180..9202)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9329..9784
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9416..9435)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9626..9647
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9810..9914
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9915..9983,9984..10775)
                     /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(10133..10152)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10946..11534
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11435..11454
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
       61 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      121 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      181 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
      241 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      301 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      361 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct
      421 tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa
      481 ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta
      541 ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc
      601 agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag
      661 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
      721 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
      781 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
      841 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
      901 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
      961 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     1021 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     1081 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     1141 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     1201 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     1261 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     1321 cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     1381 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     1441 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     1501 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     1561 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     1621 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     1681 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     1741 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     1801 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     1861 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     1921 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     1981 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     2041 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     2101 ctcccaaccc cgaggggacc cagagagggc ctatttccca tgattccttc atatttgcat
     2161 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     2221 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     2281 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     2341 gctttatata tcttgtggaa aggacgaaac accggagaag gacaggcagt gtcggtttta
     2401 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     2461 gagtcggtgc ttttttgaat tctagatctt gagacaaatg gcagtattca tccacaattt
     2521 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     2581 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     2641 ttattacagg gacagcagag atccactttg gcgccggctc gagtggctcc ggtgcccgtc
     2701 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     2761 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     2821 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     2881 ttctttttcg caacgggttt gccgccagaa cacaggtgtc gtgacgcggg atccgccacc
     2941 atggattaca aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc
     3001 cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct
     3061 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
     3121 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
     3181 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
     3241 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     3301 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     3361 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     3421 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     3481 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     3541 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     3601 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     3661 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     3721 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     3781 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     3841 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     3901 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     3961 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     4021 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     4081 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
     4141 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     4201 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     4261 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     4321 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     4381 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
     4441 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
     4501 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
     4561 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
     4621 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
     4681 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
     4741 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
     4801 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
     4861 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
     4921 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
     4981 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
     5041 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
     5101 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
     5161 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     5221 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     5281 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     5341 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     5401 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     5461 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     5521 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     5581 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     5641 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     5701 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     5761 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     5821 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     5881 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     5941 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     6001 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     6061 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     6121 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     6181 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     6241 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     6301 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
     6361 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
     6421 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     6481 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     6541 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     6601 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     6661 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     6721 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     6781 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     6841 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     6901 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     6961 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
     7021 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     7081 tacgagacac ggatcgacct gtctcagctg ggaggcgaca agcgtcctgc tgctactaag
     7141 aaagctggtc aagctaagaa aaagaaagct agcggcagcg gcgccaccaa cttcagcctg
     7201 ctgaagcagg ccggcgacgt ggaggagaac cccggcccca tgaccgagta caagcccacg
     7261 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     7321 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     7381 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     7441 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     7501 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     7561 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     7621 gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     7681 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     7741 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     7801 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa
     7861 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     7921 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     7981 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     8041 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     8101 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     8161 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     8221 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     8281 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     8341 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     8401 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa
     8461 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     8521 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
     8581 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     8641 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     8701 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta
     8761 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     8821 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     8881 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     8941 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     9001 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     9061 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     9121 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     9181 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     9241 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     9301 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     9361 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     9421 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     9481 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     9541 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     9601 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     9661 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     9721 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     9781 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     9841 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     9901 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     9961 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
    10021 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
    10081 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
    10141 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
    10201 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
    10261 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
    10321 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
    10381 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
    10441 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
    10501 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
    10561 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
    10621 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
    10681 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
    10741 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
    10801 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
    10861 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
    10921 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
    10981 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
    11041 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
    11101 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
    11161 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
    11221 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
    11281 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
    11341 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
    11401 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
    11461 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
    11521 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
    11581 cttttgctca
//
