LOCUS       sequence-429465-       11590 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Lentiviral vector expressing Cas9 and an sgRNA targeting human 
            MTHFD1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11590)
  AUTHORS   Flickinger KM, Wilson KM, Rossiter NJ, Hunger AL, Vishwasrao PV, Lee
            TD, Mellado Fritz CA, Richards RM, Hall MD, Cantor JR
  TITLE     Conditional lethality profiling reveals anticancer mechanisms of 
            action and drug-nutrient interactions.
  JOURNAL   Sci Adv. 2024 Oct 4;10(40):eadq3591. doi: 10.1126/sciadv.adq3591. 
            Epub 2024 Oct 4.
  PUBMED    39365851
REFERENCE   2  (bases 1 to 11590)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2024-10-4"; volume: "10(40):eadq3591. doi"; pages: " 
            10.1126/sciadv.adq3591. Epub 2024 Oct 4"
COMMENT     Sequence Label: pLentiCRISPR-v1-sgMTHFD1
FEATURES             Location/Qualifiers
     source          1..11590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1987..2062
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2110..2227
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2296..2507
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2527..2536
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2536..2559
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2566..2586
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2611..6711
                     /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             2611..6711
                     /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             6712..6759
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6775..6831
                     /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             6832..7431
                     /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(6832..6851)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7328..7348
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7447..8035
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7500..7520)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7918..7929)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8107..8340
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8418..8539
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8455..8474)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8509..8528
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8579..8714
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8734..8753)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8735..8753)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8763..8780)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8763..8779)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8772..8794)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8921..9376
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9008..9027)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9218..9239
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9402..9506
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9507..9575,9576..10367)
                     /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(9725..9744)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10538..11126
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11027..11046
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11280..11297
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11414..11435
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11450..11455,11456..11473,11474..11480)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11488..11504
                     /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     11493..11515
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11512..11528
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11512..11528
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11547..11567
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11549..11567
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgcaga agcaagtcat
     1981 gcatcggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     2041 aaaagtggca ccgagtcggt gcttttttga attctagatc ttgagacaaa tggcagtatt
     2101 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     2161 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     2221 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagtggct
     2281 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     2341 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     2401 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     2461 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg
     2521 ggatccgcca ccatggatta caaagacgat gacgataaga tggccccaaa gaagaagcgg
     2581 aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc
     2641 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     2701 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     2761 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     2821 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     2881 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     2941 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     3001 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     3061 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     3121 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     3181 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     3241 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     3301 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
     3361 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     3421 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     3481 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     3541 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     3601 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     3661 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     3721 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     3781 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     3841 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     3901 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     3961 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     4021 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     4081 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     4141 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     4201 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     4261 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     4321 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     4381 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     4441 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     4501 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     4561 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     4621 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     4681 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     4741 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     4801 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     4861 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     4921 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     4981 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     5041 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     5101 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     5161 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     5221 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     5281 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     5341 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     5401 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     5461 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     5521 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     5581 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     5641 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     5701 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     5761 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     5821 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     5881 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     5941 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     6001 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     6061 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     6121 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     6181 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     6241 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     6301 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     6361 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     6421 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     6481 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     6541 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     6601 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     6661 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caagcgtcct
     6721 gctgctacta agaaagctgg tcaagctaag aaaaagaaag ctagcggcag cggcgccacc
     6781 aacttcagcc tgctgaagca ggccggcgac gtggaggaga accccggccc catgaccgag
     6841 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     6901 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     6961 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     7021 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     7081 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     7141 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     7201 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     7261 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     7321 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     7381 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa
     7441 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     7501 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     7561 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     7621 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     7681 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     7741 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     7801 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     7861 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     7921 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     7981 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
     8041 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     8101 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
     8161 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     8221 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     8281 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     8341 gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     8401 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     8461 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     8521 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     8581 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     8641 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     8701 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     8761 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     8821 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     8881 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     8941 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     9001 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     9061 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     9121 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     9181 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     9241 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     9301 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     9361 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     9421 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     9481 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     9541 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     9601 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     9661 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     9721 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     9781 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     9841 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     9901 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     9961 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
    10021 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
    10081 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
    10141 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
    10201 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
    10261 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
    10321 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
    10381 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
    10441 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
    10501 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
    10561 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
    10621 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
    10681 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
    10741 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
    10801 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
    10861 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
    10921 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
    10981 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
    11041 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
    11101 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
    11161 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
    11221 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
    11281 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
    11341 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
    11401 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
    11461 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
    11521 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
    11581 agctgcaagc
//
