LOCUS       sequence-271995-       14962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for expression of high-specificity eSpCas9(1.1) 
            and sgRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14962)
  AUTHORS   Bauer M, Flatt JW, Seiler D, Cardel B, Emmenlauer M, Boucke K, 
            Suomalainen M, Hemmi S, Greber UF
  TITLE     The E3 Ubiquitin Ligase Mind Bomb 1 Controls Adenovirus Genome 
            Release at the Nuclear Pore Complex.
  JOURNAL   Cell Rep. 2019 Dec 17;29(12):3785-3795.e8. doi: 
            10.1016/j.celrep.2019.11.064.
  PUBMED    31851912
REFERENCE   2  (bases 1 to 14962)
  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.celrep.2019.11"; journalName: "Cell Rep"; date: 
            "2019-12-17- 17"; volume: "29"; issue: "12"; pages: "3785-3795.e8"
COMMENT     Sequence Label: Lenti-eCas9
FEATURES             Location/Qualifiers
     source          1..14962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2608..2856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2608..2628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2779..2798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4738..4813
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4875..5086
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5101..5110
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5110..5175
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5182..5202
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(5227..7764,7765..7767,7768..8229,8230..8232,8233..8400,
                     8401..8403,8404..9327)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             9328..9375
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9385..9441
                     /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             9442..10038
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     9935..9955
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    10054..10642
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10107..10127)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10525..10536)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10714..10947
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10973..10990)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10979..11203
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11249..11677
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11336..11355)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11546..11567
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11686..11706)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11691..12020
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11871..12006
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11933..11952
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12068..12115
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12134..12508
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    12638..12759
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12675..12694)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12729..12748
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12808..12824)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12808..12824)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12821..12843)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12832..12848
                     /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)."
     promoter        complement(join(12856..12862,12863..12880,12881..12886))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12901..12922
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13039..13056)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13210..13798)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13290..13309)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13969..14760,14761..14829))
                     /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     14592..14611
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14830..14934)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacaccggag acggttgtaa atgagcacac
     2881 aaaatacaca tgctaaaata ttatattcta tgacctttat aaaatcaacc aaaatcttct
     2941 ttttaataac tttagtatca ataattagaa tttttatgtt cctttttgca aacttttaat
     3001 aaaaatgagc aaaataaaaa aacgctagtt ttagtaactc gcgttgtttt cttcaccttt
     3061 aataatagct actccaccac ttgttcctaa gcggtcagct cctgcttcaa tcattttttg
     3121 agcatcttca aatgttctaa ctccaccagc tgctttaact aaagcattgt ctttaacaac
     3181 tgacttcatt agtttaacat cttcaaatgt tgcacctgat tttgaaaatc ctgttgatgt
     3241 tttaacaaat tctaatccag cttcaacagc tatttcacaa gctttcatga tttcttcttt
     3301 tgttaataaa caattttcca taatacattt aacaacatgt gatccagctg ctttttttac
     3361 agctttcatg tcttctaaaa ctaattcata atttttgtct tttaatgcac caatatttaa
     3421 taccatatca atttctgttg caccatcttt aattgcttca gaaacttcga atgcttttgt
     3481 agctgttgtg catgcaccta gaggaaaacc tacaacattt gttattccta catttgtgcc
     3541 ttttaataat tctttacaat agcttgttca atatgaatta acacaaactg ttgcaaaatc
     3601 aaattcaatt gcttcatcac ataattgttt aatttcagct ttcgtagcat cttgttttaa
     3661 taatgtgtga tctatatatt tgtttagttt cattttttct cctatatatt catttttaat
     3721 tttaattctt taataatttc gtctacttta actttagcgt tttgaacaga ttcaccaaca
     3781 cctataaaat aaatttttag tttaggttca gttccacttg ggcgaacagc aaatcatgac
     3841 ttatcttcta aataaaattt tagtaagtct tgtcctggca tattatacat tccatcgatg
     3901 tagtcttcaa cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat
     3961 gatttcatta atggttcaat ttttaatttc ttttcttctg gtttaaaatt caagtttaaa
     4021 gtgaaagtgt aatatgcacc catttcttta aataaatctt ctaaatagtc tactaatgtt
     4081 ttattttgtt ttttataaaa tcaagcagcc tctgctatta atatagaagc ttgtattcca
     4141 tctttatctc tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca
     4201 aaatttaatc cgttatcttc ttctttagca atttctctac ccattcattt aaatccagtt
     4261 aaagttttta caatattaac tccatatttt tcatgagcga ttctatcacc caaatcactt
     4321 gttacaaaac ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt
     4381 gataattttc aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga
     4441 ccatcatgtt ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata
     4501 tctgcatcat gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga
     4561 tttggattta caacattttt aaatgtttca tcttcaaatg catgctcttc aacctcaata
     4621 acgttatatc ctgattcacg taatattttt ggggtaaatt tagttcctgt tccattaact
     4681 gcgctaaaaa taatttttaa atctttttta gcttcttgct cttttttgta cgtctctgtt
     4741 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     4801 accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc
     4861 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     4921 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     4981 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     5041 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagat
     5101 gccaccatgg actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa
     5161 gacgatgacg ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca
     5221 gcagccgaca agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc
     5281 gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac
     5341 cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc
     5401 gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc
     5461 tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac
     5521 agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc
     5581 ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga
     5641 aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc
     5701 cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc
     5761 gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac
     5821 cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc
     5881 agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga
     5941 aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc
     6001 gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg
     6061 gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc
     6121 atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc
     6181 tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg
     6241 cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac
     6301 gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc
     6361 ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg
     6421 cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg
     6481 cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag
     6541 atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac
     6601 agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag
     6661 gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat
     6721 aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc
     6781 gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc
     6841 ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg
     6901 accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa
     6961 atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa
     7021 attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc
     7081 gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat
     7141 gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg
     7201 ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc
     7261 ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac
     7321 gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc
     7381 ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag
     7441 acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc
     7501 gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag
     7561 agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac
     7621 cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg
     7681 cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac
     7741 catatcgtgc ctcagagctt tctggccgac gactccatcg acaacaaggt gctgaccaga
     7801 agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg
     7861 aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat
     7921 ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga
     7981 cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg
     8041 aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag
     8101 tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac
     8161 aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa
     8221 aagtaccctg cgctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg
     8281 aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac
     8341 agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag
     8401 gcgcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat
     8461 tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag
     8521 gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg
     8581 atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg
     8641 gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt
     8701 gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc
     8761 gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct
     8821 aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa
     8881 ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc
     8941 agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg
     9001 gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga
     9061 gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat
     9121 aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga
     9181 gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc
     9241 aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg
     9301 atcgacctgt ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag
     9361 gcaaaaaaga aaaagggatc cggcgcaaca aacttctctc tgctgaaaca agccggagat
     9421 gtcgaagaga atcctggacc gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     9481 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     9541 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     9601 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     9661 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     9721 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     9781 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc gcccgaccac
     9841 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     9901 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     9961 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
    10021 cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac ctctggatta caaaatttgt
    10081 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
    10141 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
    10201 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
    10261 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
    10321 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
    10381 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
    10441 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
    10501 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
    10561 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
    10621 tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag
    10681 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
    10741 gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg
    10801 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
    10861 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
    10921 tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc agcctcgact
    10981 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
    11041 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
    11101 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
    11161 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
    11221 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
    11281 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
    11341 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
    11401 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
    11461 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
    11521 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
    11581 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
    11641 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
    11701 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
    11761 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
    11821 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
    11881 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
    11941 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
    12001 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
    12061 agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
    12121 gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt
    12181 cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga
    12241 cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca
    12301 ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc
    12361 cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga
    12421 gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt
    12481 gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt ccaccgccgc
    12541 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
    12601 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
    12661 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
    12721 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
    12781 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
    12841 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
    12901 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
    12961 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
    13021 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
    13081 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
    13141 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
    13201 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
    13261 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
    13321 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
    13381 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
    13441 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
    13501 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
    13561 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
    13621 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
    13681 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
    13741 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
    13801 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
    13861 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
    13921 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
    13981 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
    14041 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
    14101 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
    14161 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
    14221 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
    14281 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
    14341 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
    14401 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
    14461 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
    14521 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
    14581 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
    14641 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
    14701 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
    14761 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
    14821 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
    14881 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
    14941 tccccgaaaa gtgccacctg ac
//
