LOCUS       sequence-413636-       13052 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing doxycycline-inducible human 
            codon-optimized adenine base editor where TadA8e (ABE8e) is fused to
            the N-terminus of SpCas9(D10A)-6xNLS .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13052)
  AUTHORS   Xiao MS, Damodaran AP, Kumari B, Dickson E, Xing K, On TA, Parab N, 
            King HE, Perez AR, Guiblet WM, Duncan G, Che A, Chari R, Andresson 
            T, Vidigal JA, Weatheritt RJ, Aregger M, Gonatopoulos-Pournatzis T
  TITLE     Genome-scale exon perturbation screens uncover exons critical for 
            cell fitness.
  JOURNAL   Mol Cell. 2024 Jul 11;84(13):2553-2572.e19. doi: 
            10.1016/j.molcel.2024.05.024. Epub 2024 Jun 24.
  PUBMED    38917794
REFERENCE   2  (bases 1 to 13052)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2024.05.024"; journalName: "Mol Cell"; date: 
            "2024-07-11- 11"; volume: "84"; issue: "13"; pages: "2553-2572.e19"
COMMENT     Sequence Label: pLenti-nSpCas9-TadA8e
FEATURES             Location/Qualifiers
     source          1..13052
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..86
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    287..404
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        469..783
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    477..495
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    513..531
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    548..566
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    584..602
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    620..638
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    655..673
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    691..709
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     756..780
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             806..829
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             830..856
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1466..1489,1490..1492,1493..5566)
                     /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             1466..5566
                     /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             5588..5614
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5624..5650
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5660..5686
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5696..5722
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5732..5758
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5768..5794
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     regulatory      5804..5813
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        5813..6024
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             6040..6435
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             6442..6495
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(6496..7113,7114..7119,7120..7155,7156..7158,7159..7194,
                     7195..7197,7198..7233,7234..7242)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    7258..7846
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7311..7331)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7729..7740)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7918..8151
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8229..8350
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8266..8285)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8320..8339
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8390..8525
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8545..8564)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8546..8564)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8574..8591)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8574..8590)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8583..8605)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8732..9187
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8819..8838)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9029..9050
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9213..9317
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9318..9386,9387..10178)
                     /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(9536..9555)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10349..10937
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10838..10857
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11091..11108
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11225..11246
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11261..11266,11267..11284,11285..11291)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11299..11315
                     /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     11304..11326
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11323..11339
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11323..11339
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11358..11378
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11360..11378
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        11406..11632
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             11633..11813
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11860..11985
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12478..12711
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12896..12940
                     /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"
ORIGIN
        1 gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
       61 ccagagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
      121 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
      181 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
      241 atcataaatt ctagatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
      301 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
      361 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
      421 acagcagaga tccactttgg cgccggctcg agcgcgaatc gatatgtcga gtttactccc
      481 tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgatgtcg
      541 agtttactcc ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga
      601 gaacgtatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttatcccta
      661 tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga
      721 ggtaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc
      781 gcctggagaa ttggctagca ccatggatta caaagacgat gacgataagt acccatacga
      841 tgtgccagat tacgccagcg cttctgaggt ggagttttcc cacgagtact ggatgagaca
      901 tgccctgacc ctggccaaga gggcacggga tgagagggag gtgcctgtgg gagccgtgct
      961 ggtgctgaac aatagagtga tcggcgaggg ctggaacaga gccatcggcc tgcacgaccc
     1021 aacagcccat gccgaaatta tggccctgag acagggcggc ctggtcatgc agaactacag
     1081 actgattgac gccaccctgt acgtgacatt cgagccttgc gtgatgtgcg ccggcgccat
     1141 gatccactct aggatcggcc gcgtggtgtt tggcgtgagg aactcaaaaa gaggcgccgc
     1201 aggctccctg atgaacgtgc tgaactaccc cggcatgaat caccgcgtcg aaattaccga
     1261 gggaatcctg gcagatgaat gtgccgccct gctgtgcgat ttctatcgga tgcctagaca
     1321 ggtgttcaat gctcagaaga aggcccagag ctccatcaac tccggaggat ctagcggagg
     1381 atcctctggc agcgagacac caggaacaag cgagtcagca acaccagaga gcagtggcgg
     1441 cagcagcggg gggtcagcta gcgctgacaa gaagtacagc atcggcctgg ccatcggcac
     1501 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     1561 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
     1621 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     1681 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
     1741 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     1801 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     1861 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     1921 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     1981 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     2041 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     2101 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     2161 gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     2221 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     2281 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     2341 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     2401 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     2461 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     2521 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     2581 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     2641 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     2701 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     2761 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     2821 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     2881 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     2941 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     3001 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     3061 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt
     3121 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
     3181 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
     3241 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
     3301 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
     3361 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
     3421 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
     3481 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
     3541 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
     3601 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
     3661 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
     3721 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
     3781 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
     3841 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
     3901 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
     3961 gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga
     4021 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga
     4081 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
     4141 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
     4201 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
     4261 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
     4321 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
     4381 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
     4441 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
     4501 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
     4561 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
     4621 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
     4681 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
     4741 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
     4801 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
     4861 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
     4921 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
     4981 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
     5041 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
     5101 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
     5161 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
     5221 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
     5281 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
     5341 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
     5401 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
     5461 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
     5521 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggtgacagcg gcgggaattc
     5581 cagatcccct gctgctaaac gtgttaagct tgatgggggt agcccggcag ccaagagagt
     5641 caaactcgac ggcggctccc cagcggcaaa aagggtgaaa ctagacgggg gtagccccgc
     5701 cgcgaagcgt gtaaagctgg atggaggatc gcctgcggct aagcgagtca aattagacgg
     5761 tgggtcaccg gctgcaaagc gcgtcaaatt ggattgagct agcgccacca tggggcagag
     5821 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc
     5881 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     5941 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     6001 aacgggtttg ccgccagaac acagttcgaa caattcacca tggccaagcc tttgtctcaa
     6061 gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa
     6121 gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat
     6181 gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct
     6241 gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg
     6301 agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata
     6361 gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt
     6421 tatgtgtggg agggcggtac cgaaggtaga ggttctctcc tcacttgtgg tgatgttgaa
     6481 gaaaaccctg gtccaatgtc tagactggac aagagcaaag tcataaacgg agctctggaa
     6541 ttactcaatg gtgtcggtat cgaaggcctg acgacaagga aactcgctca aaagctggga
     6601 gttgagcagc ctaccctgta ctggcacgtg aagaacaagc gggccctgct cgatgccctg
     6661 ccaatcgaga tgctggacag gcatcatacc cacttctgcc ccctggaagg cgagtcatgg
     6721 caagactttc tgcggaacaa cgccaagtca taccgctgtg ctctcctctc acatcgcgac
     6781 ggggctaaag tgcatctcgg cacccgccca acagagaaac agtacgaaac cctggaaaat
     6841 cagctcgcgt tcctgtgtca gcaaggcttc tccctggaga acgcactgta cgctctgtcc
     6901 gccgtgggcc actttacact gggctgcgta ttggaggaac aggagcatca agtagcaaaa
     6961 gaggaaagag agacacctac caccgattct atgcccccac ttctgagaca agcaattgag
     7021 ctgttcgacc ggcagggagc cgaacctgcc ttccttttcg gcctggaact aatcatatgt
     7081 ggcctggaga aacagctaaa gtgcgaaagc ggcgggccga ccgacgccct tgacgatttt
     7141 gacttagaca tgctcccagc cgatgccctt gacgactttg accttgatat gctgcctgct
     7201 gacgctcttg acgattttga ccttgacatg ctccccgggt aaacgcgtta agtcgacaat
     7261 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     7321 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     7381 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     7441 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     7501 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     7561 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     7621 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     7681 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     7741 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     7801 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac
     7861 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     7921 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     7981 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     8041 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     8101 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata
     8161 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     8221 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     8281 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     8341 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
     8401 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     8461 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     8521 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     8581 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     8641 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     8701 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     8761 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     8821 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     8881 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     8941 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     9001 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     9061 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     9121 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     9181 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     9241 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     9301 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     9361 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     9421 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     9481 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     9541 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     9601 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     9661 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     9721 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     9781 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     9841 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     9901 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     9961 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
    10021 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
    10081 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
    10141 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
    10201 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
    10261 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
    10321 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
    10381 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
    10441 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
    10501 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
    10561 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
    10621 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
    10681 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
    10741 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
    10801 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
    10861 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
    10921 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
    10981 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
    11041 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
    11101 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
    11161 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
    11221 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
    11281 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
    11341 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag
    11401 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
    11461 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
    11521 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
    11581 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
    11641 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
    11701 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
    11761 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
    11821 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
    11881 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
    11941 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
    12001 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
    12061 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
    12121 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
    12181 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
    12241 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
    12301 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
    12361 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
    12421 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
    12481 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
    12541 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
    12601 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
    12661 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
    12721 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
    12781 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
    12841 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
    12901 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
    12961 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
    13021 aatagttttt gctgtacttt ctatagtgaa ta
//
