LOCUS       sequence-459736-       11761 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express E14-ABE in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11761)
  AUTHORS   Zhao N, Zhou J, Tao T, Wang Q, Tang J, Li D, Gou S, Guan Z, Olajide 
            JS, Lin J, Wang S, Li X, Zhou J, Gao Z, Wang G
  TITLE     Evolved cytidine and adenine base editors with high precision and 
            minimized off-target activity by a continuous directed evolution 
            system in mammalian cells.
  JOURNAL   Nat Commun. 2024 Sep 17;15(1):8140. doi: 10.1038/s41467-024-52483-3.
  PUBMED    39289397
REFERENCE   2  (bases 1 to 11761)
  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 
            Commun."; date: "2024-09-17"; volume: "15(1):8140. doi"; pages: " 
            10.1038/s41467-024-52483-3"
COMMENT     Sequence Label: Lenti-E14-ABE-Cas9n-puro
FEATURES             Location/Qualifiers
     source          1..11761
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        17..243
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             244..424
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    471..596
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1089..1322
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1507..1551
                     /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    1735..1852
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1921..2132
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2194..2214
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2809..2832,2833..2835,2836..6909)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2809..6909
                     /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
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6922..6942
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6958..7014
                     /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             7015..7614
                     /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(7015..7034)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7511..7531
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7630..8218
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7683..7703)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8101..8112)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8290..8523
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8601..8722
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8638..8657)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8692..8711
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8762..8897
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8917..8936)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8918..8936)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8946..8963)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8946..8962)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8955..8977)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9104..9559
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9191..9210)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9401..9422
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9585..9689
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9690..9758,9759..10550)
                     /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(9908..9927)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10721..11309
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11210..11229
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11463..11480
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11597..11618
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11633..11638,11639..11656,11657..11663)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11671..11687
                     /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     11676..11698
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11695..11711
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11695..11711
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11730..11750
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11732..11750
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg
       61 atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa
      121 gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga
      181 cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata
      241 aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      301 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      361 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
      421 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
      481 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
      541 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
      601 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
      661 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
      721 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
      781 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
      841 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
      901 agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga
      961 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     1021 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     1081 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     1141 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     1201 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     1261 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     1321 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     1381 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     1441 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     1501 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     1561 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     1621 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     1681 tcaccattat cgtttcagac ccacctccca accccgaggg gacccagacc acaattttaa
     1741 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     1801 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     1861 ttacagggac agcagagatc cactttggcg ccggctcgag tggctccggt gcccgtcagt
     1921 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     1981 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     2041 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     2101 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc cgccaccatg
     2161 aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa agtctctgag
     2221 gtggagtttt cccacgagta ctggatgaga catgccctga ccctggccaa gagggcacgg
     2281 gatgagaggg aggtgcctgt gggagccgtg ctggtgctga acggtggagt gatcggcgag
     2341 ggctggaaca gagccatcgg cctgcacgac ccaacagccc atgccgaaat tatggccctg
     2401 agacagggcg gcctggtcat gcagaactac agactgattg acgccaccct gtacgtgaca
     2461 ttcgagcctt gcgtgatgtg cgccggcgcc atgatccact ctaggatctg ccgcgtggtg
     2521 tttggatggg gaaattctaa aagaggcgcc gcaggctccc tgatgaacgt gctgaactac
     2581 cccggcatga atcaccgcgt cgaaattacc gagggaatcc tggcagatga atgtgccgcc
     2641 ctgctgtgcg atttctatcg gatgcctaga caggtgttca atgctcagaa gaaggcccag
     2701 agctccatca actccggagg atctggcgga ggctcctctg gctctgagac acctggcaca
     2761 agcgagagcg caacacctga aagcagcggg ggcagcagcg gggggtcaga caagaagtac
     2821 agcatcggcc tggccatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     2881 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     2941 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     3001 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     3061 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     3121 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     3181 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     3241 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     3301 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     3361 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     3421 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     3481 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     3541 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     3601 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     3661 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     3721 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     3781 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     3841 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     3901 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     3961 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     4021 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     4081 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     4141 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     4201 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     4261 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     4321 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     4381 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     4441 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     4501 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     4561 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     4621 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     4681 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     4741 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     4801 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     4861 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     4921 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     4981 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     5041 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     5101 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     5161 gagggcatca aagagctggg cagccagatc ctgaaagaac acccagtgga aaacacccag
     5221 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     5281 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     5341 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     5401 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     5461 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     5521 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     5581 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     5641 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     5701 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     5761 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     5821 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     5881 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     5941 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     6001 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     6061 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     6121 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     6181 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     6241 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     6301 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     6361 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     6421 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     6481 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     6541 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     6601 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     6661 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     6721 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     6781 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     6841 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
     6901 ggaggcgatt caggcggatc tcccaagaag aagaggaaag tcgctagcgg cagcggcgcc
     6961 accaacttca gcctgctgaa gcaggccggc gacgtggagg agaaccccgg ccccatgacc
     7021 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     7081 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     7141 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     7201 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     7261 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     7321 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     7381 tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     7441 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     7501 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     7561 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaacgcgt
     7621 taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     7681 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     7741 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     7801 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     7861 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     7921 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     7981 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     8041 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     8101 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     8161 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgt
     8221 cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     8281 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta
     8341 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     8401 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     8461 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     8521 gcagtacgta tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     8581 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     8641 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     8701 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     8761 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     8821 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     8881 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     8941 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     9001 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     9061 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     9121 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     9181 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     9241 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     9301 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     9361 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     9421 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     9481 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     9541 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     9601 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     9661 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     9721 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     9781 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     9841 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     9901 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     9961 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
    10021 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
    10081 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
    10141 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
    10201 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
    10261 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
    10321 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
    10381 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
    10441 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
    10501 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
    10561 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
    10621 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
    10681 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
    10741 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
    10801 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
    10861 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
    10921 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
    10981 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
    11041 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
    11101 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
    11161 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
    11221 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
    11281 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
    11341 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
    11401 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
    11461 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
    11521 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
    11581 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
    11641 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
    11701 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
    11761 t
//
