LOCUS       sequence-200755-       12376 bp    DNA     circular SYN 14-APR-2025
DEFINITION  SaCas9 expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12376)
  AUTHORS   Boettcher M, Tian R, Blau JA, Markegard E, Wagner RT, Wu D, Mo X, 
            Biton A, Zaitlen N, Fu H, McCormick F, Kampmann M, McManus MT
  TITLE     Dual gene activation and knockout screen reveals directional 
            dependencies in genetic networks.
  JOURNAL   Nat Biotechnol. 2018 Jan 15. pii: nbt.4062. doi: 10.1038/nbt.4062.
  PUBMED    29334369
REFERENCE   2  (bases 1 to 12376)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2018 Jan 15. pii: nbt.4062. doi: 10.1038/nbt.4062."
COMMENT     Sequence Label: pMB-SaCas9
FEATURES             Location/Qualifiers
     source          1..12376
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        247..626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        628..826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             844..1024
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1071..1196
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1695..1928
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2113..2157
                     /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    2455..2572
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     polyA_signal    2760..2894
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2797..2816)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4306..4359)
                     /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             complement(4369..5391)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4369..5391)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(5468..5492)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(5489..5527)
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        5572..5783
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5801..5810
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5813..5833
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5858..9013
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             9014..9061
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9068..9148
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     protein_bind    complement(9176..9209)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    9265..9853
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9318..9338)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9736..9747)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9856..9872)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9857..9873)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10382..10562
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(10624..11212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10704..10723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11383..12174,12175..12243))
                     /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     12006..12025
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12244..12348)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcttaagcgg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt
      841 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      901 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      961 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     1021 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
     1081 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
     1141 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
     1201 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
     1261 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     1321 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     1381 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     1441 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     1501 agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat cttcagacct
     1561 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1621 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1681 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1741 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1801 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1861 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1921 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     1981 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     2041 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     2101 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     2161 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     2221 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     2281 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     2341 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     2401 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     2461 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2521 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2581 ttacagggac agcagagatc cagtttggtt agtaccgggc cctagagatc acgagactag
     2641 cctcgagaga tctgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     2701 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     2761 acttgtttat tgcagcttat aatggttaca aataaggcaa tagcatcaca aatttcacaa
     2821 ataaggcatt tttttcactg cattctagtt ttggtttgtc caaactcatc aatgtatctt
     2881 atcatgtctg gatctcaaat ccctcggaag ctgcgcctgt catcgaattc ctgcagcccg
     2941 gtgcatgact aagctagtac cggttaggat gcatgctagc tcagttagcc tcccccatct
     3001 ctcgacgcgg ccgcttcact ggcgctgttg gtgccggcac ctgacacagc agaggatgca
     3061 gagcccaagg aaacccagaa agccgaagga gccacccagc acgcaagcca ggaacactgt
     3121 ctggttcacc cctctggata aaacctggat cctggagtcc atcttgacct tatcaccttc
     3181 actcagtaga cactgccaca gccctgtctc aggggccacc acttgaacta ctttctgctc
     3241 ctcagagacc ctggcctcct ggttctcctg cttcagggtc agtctcatct tgggagaggt
     3301 aggtcccatc acctcacagg tcaaagtatt gttgagctga gccactttca tcaccaccag
     3361 gttcacttcc tgatgcagtg tccctttgtc cagagtcaga gtcaggttgc cagaaccagc
     3421 aaactgaagc gagacctggg gtatcttgag ggtgagtggg agcgtttcct tcagctggag
     3481 cttgaggtct ttggtggact tttgtacgga cacctctttg ttctttatgg agaaggagat
     3541 ccagggctgg aagaaagaat ccttctctgc cttccacatc agctctcccc acccgttttc
     3601 ctctgcaaag ttgagtggga aggagaactc cgctgactct ccctcactct tataggccgt
     3661 gatagctgtg ctctgaaaac ccagcactga gagtgtcatg ccgaaccagt tctttttctg
     3721 gtccagggtc acggtgcagt tccagaagtc gctgtcctga acccttaggt tggacatgga
     3781 gagaactttg gaaccactga caactttacc ctttttgtgt ttgcactctg tcaaggggtt
     3841 agagacctta gagttgctat ccaaggtcag ggtcaggctc tgcccttgca acaggctggt
     3901 acccggactg aaggtcactt tgaacaccca caactccacc tcctctttcc tgttctccag
     3961 ctcacagata taagtctgag agtcttccat cttaagttta ttgatgatga gaggaaacga
     4021 tcctttctcc catgcccctt ttttggaatc aaaacgatca aactgcgaag gcgaacctcc
     4081 tctaattaat acacctttgc catgctgccc cagaatcttc ctctggtcag agaacttcca
     4141 ggtgaagact gtgatcttct tctgggaact ctcgcagggc agttctgctg attccccttc
     4201 cttccccagc accagcgtct tcccttgagt gacagctagg agttgtgaca gctgcagcag
     4261 cagcagcagc aagcgcctaa gagagatggc tcggcacatg tccggtgggc caggattctc
     4321 ctcgacgtca ccgcatgtta gcagacttcc tctgccctcg gcgcgccctt cctttgccct
     4381 cggacgagtg ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt
     4441 ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg
     4501 gacgattgcg tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa
     4561 gctctgatag agttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc
     4621 tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca
     4681 cggcctccag aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct
     4741 ccagtcaatg accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc
     4801 cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag
     4861 agcctgcgcg acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg
     4921 gggatcagca atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg
     4981 tccgaatggg ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatggc
     5041 ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac
     5101 accctgtgca cggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag
     5161 cacttccgga atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta
     5221 gaaaccatcg gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct
     5281 gaaagcacga gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt
     5341 ttcgatcaga aacttctcga cagacgtcgc ggtgagttca ggctttttca tggtggctcg
     5401 agcggaggct ggatcggtcc cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc
     5461 tccaggcgat ctgacggttc actaaacgag ctctgcttat ataggcctcc caccgtacac
     5521 gcctaccctc gagaagcttg atatctctag agctctagtg tgcccgtcag tgggcagagc
     5581 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
     5641 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     5701 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     5761 acgggtttgc cgccagaaca gtgagctagc gctaccggtc gccaccatgg ccccaaagaa
     5821 gaagcggaag gtcggtatcc acggagtccc agcagccaag cggaactaca tcctgggcct
     5881 ggacatcggc atcaccagcg tgggctacgg catcatcgac tacgagacac gggacgtgat
     5941 cgatgccggc gtgcggctgt tcaaagaggc caacgtggaa aacaacgagg gcaggcggag
     6001 caagagaggc gccagaaggc tgaagcggcg gaggcggcat agaatccaga gagtgaagaa
     6061 gctgctgttc gactacaacc tgctgaccga ccacagcgag ctgagcggca tcaaccccta
     6121 cgaggccaga gtgaagggcc tgagccagaa gctgagcgag gaagagttct ctgccgccct
     6181 gctgcacctg gccaagagaa gaggcgtgca caacgtgaac gaggtggaag aggacaccgg
     6241 caacgagctg tccaccaaag agcagatcag ccggaacagc aaggccctgg aagagaaata
     6301 cgtggccgaa ctgcagctgg aacggctgaa gaaagacggc gaagtgcggg gcagcatcaa
     6361 cagattcaag accagcgact acgtgaaaga agccaaacag ctgctgaagg tgcagaaggc
     6421 ctaccaccag ctggaccaga gcttcatcga cacctacatc gacctgctgg aaacccggcg
     6481 gacctactat gagggacctg gcgagggcag ccccttcggc tggaaggaca tcaaagaatg
     6541 gtacgagatg ctgatgggcc actgcaccta cttccccgag gaactgcgga gcgtgaagta
     6601 cgcctacaac gccgacctgt acaacgccct gaacgacctg aacaatctcg tgatcaccag
     6661 ggacgagaac gagaagctgg aatattacga gaagttccag atcatcgaga acgtgttcaa
     6721 gcagaagaag aagcccaccc tgaagcagat cgccaaagaa atcctcgtga acgaagagga
     6781 tattaagggc tacagagtga ccagcaccgg caagcccgag ttcaccaacc tgaaggtgta
     6841 ccacgacatc aaggacatta ccgcccggaa agagattatt gagaacgccg agctgctgga
     6901 tcagattgcc aagatcctga ccatctacca gagcagcgag gacatccagg aagaactgac
     6961 caatctgaac tccgagctga cccaggaaga gatcgagcag atctctaatc tgaagggcta
     7021 taccggcacc cacaacctga gcctgaaggc catcaacctg atcctggacg agctgtggca
     7081 caccaacgac aaccagatcg ctatcttcaa ccggctgaag ctggtgccca agaaggtgga
     7141 cctgtcccag cagaaagaga tccccaccac cctggtggac gacttcatcc tgagccccgt
     7201 cgtgaagaga agcttcatcc agagcatcaa agtgatcaac gccatcatca agaagtacgg
     7261 cctgcccaac gacatcatta tcgagctggc ccgcgagaag aactccaagg acgcccagaa
     7321 aatgatcaac gagatgcaga agcggaaccg gcagaccaac gagcggatcg aggaaatcat
     7381 ccggaccacc ggcaaagaga acgccaagta cctgatcgag aagatcaagc tgcacgacat
     7441 gcaggaaggc aagtgcctgt acagcctgga agccatccct ctggaagatc tgctgaacaa
     7501 ccccttcaac tatgaggtgg accacatcat ccccagaagc gtgtccttcg acaacagctt
     7561 caacaacaag gtgctcgtga agcaggaaga aaacagcaag aagggcaacc ggaccccatt
     7621 ccagtacctg agcagcagcg acagcaagat cagctacgaa accttcaaga agcacatcct
     7681 gaatctggcc aagggcaagg gcagaatcag caagaccaag aaagagtatc tgctggaaga
     7741 acgggacatc aacaggttct ccgtgcagaa agacttcatc aaccggaacc tggtggatac
     7801 cagatacgcc accagaggcc tgatgaacct gctgcggagc tacttcagag tgaacaacct
     7861 ggacgtgaaa gtgaagtcca tcaatggcgg cttcaccagc tttctgcggc ggaagtggaa
     7921 gtttaagaaa gagcggaaca aggggtacaa gcaccacgcc gaggacgccc tgatcattgc
     7981 caacgccgat ttcatcttca aagagtggaa gaaactggac aaggccaaaa aagtgatgga
     8041 aaaccagatg ttcgaggaaa agcaggccga gagcatgccc gagatcgaaa ccgagcagga
     8101 gtacaaagag atcttcatca ccccccacca gatcaagcac attaaggact tcaaggacta
     8161 caagtacagc caccgggtgg acaagaagcc taatagagag ctgattaacg acaccctgta
     8221 ctccacccgg aaggacgaca agggcaacac cctgatcgtg aacaatctga acggcctgta
     8281 cgacaaggac aatgacaagc tgaaaaagct gatcaacaag agccccgaaa agctgctgat
     8341 gtaccaccac gacccccaga cctaccagaa actgaagctg attatggaac agtacggcga
     8401 cgagaagaat cccctgtaca agtactacga ggaaaccggg aactacctga ccaagtactc
     8461 caaaaaggac aacggccccg tgatcaagaa gattaagtat tacggcaaca aactgaacgc
     8521 ccatctggac atcaccgacg actaccccaa cagcagaaac aaggtcgtga agctgtccct
     8581 gaagccctac agattcgacg tgtacctgga caatggcgtg tacaagttcg tgaccgtgaa
     8641 gaatctggat gtgatcaaaa aagaaaacta ctacgaagtg aatagcaagt gctatgagga
     8701 agctaagaag ctgaagaaga tcagcaacca ggccgagttt atcgcctcct tctacaacaa
     8761 cgatctgatc aagatcaacg gcgagctgta tagagtgatc ggcgtgaaca acgacctgct
     8821 gaaccggatc gaagtgaaca tgatcgacat cacctaccgc gagtacctgg aaaacatgaa
     8881 cgacaagagg ccccccagga tcattaagac aatcgcctcc aagacccaga gcattaagaa
     8941 gtacagcaca gacattctgg gcaacctgta tgaagtgaaa tctaagaagc accctcagat
     9001 catcaaaaag ggcaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     9061 gggatcctac ccatacgatg ttccagatta cgcttaccca tacgatgttc cagattacgc
     9121 ttacccatac gatgttccag attacgctta aattaagaat tcgtcgaggg acctaataac
     9181 ttcgtatagc atacattata cgaagttata catgtttaag ggttccggtt ccactaggta
     9241 caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     9301 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     9361 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     9421 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     9481 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     9541 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     9601 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     9661 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     9721 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     9781 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     9841 gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac atggagcaat
     9901 cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga
     9961 ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc
    10021 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
    10081 ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga
    10141 ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta
    10201 caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag agaacacccg
    10261 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tattagagtg
    10321 gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc cggactgtac
    10381 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
    10441 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
    10501 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
    10561 cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
    10621 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
    10681 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
    10741 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
    10801 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
    10861 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
    10921 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
    10981 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
    11041 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
    11101 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
    11161 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
    11221 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
    11281 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
    11341 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
    11401 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
    11461 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
    11521 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
    11581 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
    11641 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
    11701 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
    11761 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
    11821 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
    11881 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
    11941 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
    12001 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
    12061 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
    12121 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
    12181 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
    12241 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
    12301 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
    12361 aaaagtgcca cctgac
//
