LOCUS       sequence-212230-       13030 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentivirus for constitutive expression of xCas9(3.7)-BE4Gam in 
            mammalian cells (codon optimized).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13030)
  AUTHORS   Zafra MP, Schatoff EM, Katti A, Foronda M, Breinig M, Schweitzer AY,
            Simon A, Han T, Goswami S, Montgomery E, Thibado J, Kastenhuber ER, 
            Sanchez-Rivera FJ, Shi J, Vakoc CR, Lowe SW, Tschaharganeh DF, Dow 
            LE
  TITLE     Optimized base editors enable efficient editing in cells, organoids 
            and mice.
  JOURNAL   Nat Biotechnol. 2018 Jul 3. pii: nbt.4194. doi: 10.1038/nbt.4194.
  PUBMED    29969439
REFERENCE   2  (bases 1 to 13030)
  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"; journalName: "Nat 
            Biotechnol. 2018 Jul 3. pii: nbt.4194. doi: 10.1038/nbt.4194."
COMMENT     Sequence Label: pLenti-xBE4Gam-P2A-Puro
FEATURES             Location/Qualifiers
     source          1..13030
                     /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"
     misc_feature    1723..1840
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1909..2120
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2140..2149
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2149..2172
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2179..2199
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2791..3474
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             3523..7623
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 mutant with broad PAM
                     compatibility (Hu et al., 2018)"
                     /label=xCas9-3.7
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFIQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             3523..7623
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFIQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7636..7884
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             7897..7917
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7918..8166
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             8179..8199
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8215..8271
                     /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             8272..8871
                     /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(8272..8291)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8768..8788
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8887..9475
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8940..8960)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9358..9369)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9547..9780
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    9858..9979
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9895..9914)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9949..9968
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10019..10154
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10174..10193)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10175..10193)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10203..10220)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10203..10219)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10212..10234)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10361..10816
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10448..10467)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10658..10679
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10842..10946
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10947..11015,11016..11807)
                     /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(11165..11184)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11978..12566
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12467..12486
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12720..12737
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12854..12875
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12890..12895,12896..12913,12914..12920)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12928..12944
                     /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     12933..12955
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12952..12968
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12952..12968
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     12987..13007
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        12989..13007
                     /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 cccagaccac aattttaaaa gaaaaggggg
     1741 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     1801 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     1861 cagagatcca ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca
     1921 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
     1981 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     2041 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     2101 ggtttgccgc cagaacacag gtgtcgtgac gcgggatccg ccaccatgga ttacaaagac
     2161 gatgacgata agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca
     2221 gccgcaaaac ctgcaaagag aattaaatcc gcagcagcag cctacgtgcc tcaaaaccgg
     2281 gatgccgtta tcacagatat aaaaagaatc ggtgatttgc agcgcgaagc aagccgcttg
     2341 gagaccgaaa tgaatgatgc catcgcagag atcactgaga aatttgctgc ccgcatagca
     2401 ccaatcaaga ctgacatcga gacactcagt aagggcgtgc aaggctggtg cgaggctaat
     2461 cgggacgagt tgaccaacgg ggggaaggtg aaaaccgcca atcttgtgac tggcgatgtc
     2521 tcctggcgag tgagaccacc aagcgtaagc atccgaggca tggacgctgt gatggaaaca
     2581 ttggaaaggc tcggcctgca aaggtttatc agaacaaagc aggaaataaa taaggaagcc
     2641 atcctccttg agccaaaagc cgttgctggg gtagccggaa ttactgttaa gtctggtatc
     2701 gaggatttca gtatcatacc cttcgagcag gaagccggca ttagcggaag tgaaacaccc
     2761 ggtacctcag agagcgcaac tcctgagagt agctcagaga ctggcccagt ggctgtggac
     2821 cccacattga gacggcggat cgagccccat gagtttgagg tattcttcga tccgagagag
     2881 ctccgcaagg agacctgcct gctttacgaa attaattggg ggggccggca ctccatttgg
     2941 cgacatacat cacagaacac taacaagcac gtcgaagtca acttcatcga gaagttcacg
     3001 acagaaagat atttctgtcc gaacacaagg tgcagcatta cctggtttct cagctggagc
     3061 ccatgcggcg aatgtagtag ggccatcact gaattcctgt caaggtatcc ccacgtcact
     3121 ctgtttattt acatcgcaag gctgtaccac cacgctgacc cccgcaatcg acaaggcctg
     3181 cgggatttga tctcttcagg tgtgactatc caaattatga ctgagcagga gtcaggatac
     3241 tgctggagaa actttgtgaa ttatagcccg agtaatgaag cccactggcc taggtatccc
     3301 catctgtggg tacgactgta cgttcttgaa ctgtactgca tcatactggg cctgcctcct
     3361 tgtctcaaca ttctgagaag gaagcagcca cagctgacat tctttaccat cgctcttcag
     3421 tcttgtcatt accagcgact gcccccacac attctctggg ccaccgggtt gaaaagcggc
     3481 agcgagactc ccgggacctc agagtccgcc acacccgaaa gtgacaagaa gtacagcatc
     3541 ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     3601 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     3661 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
     3721 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     3781 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     3841 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     3901 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     3961 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     4021 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     4081 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     4141 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     4201 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
     4261 ctgaccccca acttcaagag caacttcgac ctggccgagg ataccaaact gcagctgagc
     4321 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     4381 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     4441 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagct gtacgacgag
     4501 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     4561 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     4621 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     4681 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     4741 ggcatcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     4801 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     4861 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     4921 aagagcgagg aaaccatcac cccctggaac ttcgagaagg tggtggacaa gggcgcttcc
     4981 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     5041 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     5101 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgacca gaaaaaggcc
     5161 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     5221 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     5281 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     5341 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     5401 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     5461 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     5521 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     5581 ttcgccaaca gaaacttcat ccagctgatc cacgacgaca gcctgacctt taaagaggac
     5641 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     5701 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     5761 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     5821 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     5881 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     5941 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     6001 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
     6061 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
     6121 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     6181 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     6241 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     6301 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     6361 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     6421 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     6481 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     6541 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     6601 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     6661 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     6721 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     6781 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     6841 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     6901 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     6961 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     7021 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     7081 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     7141 aacggccgga agagaatgct ggcctctgcc ggcgtgctgc agaagggaaa cgaactggcc
     7201 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     7261 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     7321 gagatcatcg agcagattag cgagttctcc aagagagtga tcctggccga cgctaatctg
     7381 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     7441 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     7501 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     7561 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     7621 gattcaggcg gatctactaa tctgtcagat attattgaaa aggagaccgg taagcaactg
     7681 gttatccagg aatccatcct catgctccca gaggaggtgg aagaagtcat tgggaacaag
     7741 ccggaaagcg atatactcgt gcacaccgcc tacgacgaga gcaccgacga gaatgtcatg
     7801 cttctgacta gcgacgcccc tgaatacaag ccttgggctc tggtcataca ggatagcaac
     7861 ggtgagaaca agattaagat gctctctggt ggttctccca agaagaagag gaaagtcaca
     7921 aatctctctg acatcataga gaaggagaca gggaaacaac tcgtaataca agagtccatt
     7981 cttatgctcc ctgaggaggt ggaagaagtt atcggcaaca aaccagagag tgacattctg
     8041 gtccataccg cctacgatga aagcacagac gagaacgtta tgttgctcac ttctgacgct
     8101 ccagaataca aaccttgggc actcgtcatt caggacagca acggcgagaa caagatcaaa
     8161 atgcttagcg ggggcagccc caaaaaaaag aggaaggtcg ctagcggcag cggcgccacc
     8221 aacttcagcc tgctgaagca ggccggcgac gtggaggaga accccggccc catgaccgag
     8281 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     8341 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     8401 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     8461 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     8521 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     8581 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     8641 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     8701 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     8761 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     8821 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa
     8881 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     8941 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     9001 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     9061 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     9121 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     9181 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     9241 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     9301 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     9361 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     9421 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
     9481 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     9541 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
     9601 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     9661 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     9721 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     9781 gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     9841 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     9901 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     9961 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
    10021 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
    10081 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
    10141 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
    10201 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
    10261 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
    10321 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
    10381 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
    10441 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
    10501 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
    10561 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
    10621 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
    10681 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
    10741 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
    10801 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
    10861 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
    10921 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
    10981 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
    11041 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
    11101 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
    11161 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
    11221 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
    11281 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
    11341 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
    11401 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
    11461 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
    11521 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
    11581 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
    11641 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
    11701 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
    11761 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
    11821 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
    11881 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
    11941 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
    12001 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
    12061 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
    12121 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
    12181 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
    12241 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
    12301 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
    12361 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
    12421 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
    12481 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
    12541 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
    12601 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
    12661 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
    12721 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
    12781 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
    12841 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
    12901 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
    12961 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
    13021 agctgcaagc
//
