pCW-Cas9-Blast

LOCUS       pCW-Cas9-Blast          11684 bp ds-DNA     circular SYN 16-JUN-2021
DEFINITION  Lentiviral vector for mammalian expression of doxycycline-inducible 
            Cas9 and constitutive expression of Blasticidin S deaminaase -T2A 
            -rtTA.
ACCESSION   .
VERSION     .
KEYWORDS    pCW-Cas9-Blast
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11684)
  AUTHORS   Habashy et al
  TITLE     CRISPR/Cas9-based system for loss-of-function screening
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 11684)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 16, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..11684
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        212..526
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    220..238
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    256..274
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    291..309
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    327..345
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    363..381
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    398..416
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    434..452
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     499..523
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      543..552
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             552..617
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             624..644
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             669..4769
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4770..4817
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     promoter        4827..5337
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     5256..5274
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             5347..5742
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             5743..5796
                     /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             5797..6543
                     /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"
     primer_bind     complement(6583..6603)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7001..7012)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7217..7450
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7522..7643
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7559..7578)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7613..7632
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7683..7818
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7745..7764
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7818..7889
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7890..8750
                     /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(8108..8127)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8921..9509
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9410..9429
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9663..9680
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9797..9818
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        9833..9863
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9871..9887
                     /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     9876..9898
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9895..9911
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9895..9911
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9930..9950
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9932..9950
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        9978..10204
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             10205..10385
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    10432..10557
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11050..11283
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             11468..11512
                     /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 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac
      241 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag
      301 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
      361 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt
      421 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg
      481 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggcta
      541 gcgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca
      601 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
      661 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
      721 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
      781 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
      841 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
      901 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
      961 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     1021 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     1081 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     1141 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     1201 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     1261 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     1321 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     1381 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     1441 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     1501 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     1561 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     1621 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     1681 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     1741 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     1801 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     1861 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     1921 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     1981 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     2041 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     2101 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     2161 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     2221 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     2281 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     2341 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     2401 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     2461 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     2521 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     2581 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     2641 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     2701 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     2761 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     2821 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     2881 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     2941 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     3001 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     3061 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     3121 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     3181 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     3241 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     3301 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     3361 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     3421 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     3481 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     3541 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     3601 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     3661 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     3721 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     3781 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     3841 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     3901 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     3961 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     4021 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     4081 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     4141 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     4201 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     4261 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     4321 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     4381 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     4441 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     4501 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     4561 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     4621 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     4681 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     4741 ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc
     4801 aggcaaaaaa gaaaaagtaa ggatccgggg ttggggttgc gccttttcca aggcagccct
     4861 gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac
     4921 cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc
     4981 cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg
     5041 ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac
     5101 ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc
     5161 tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt
     5221 ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg
     5281 gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagcaa
     5341 ttcaccatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct
     5401 acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc
     5461 gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa
     5521 ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg tatcgtcgcg
     5581 atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca ggtgcttctc
     5641 gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc gacggcagtt
     5701 gggattcgtg aattgctgcc ctctggttat gtgtgggagg gcgaaggtag aggttctctc
     5761 ctcacttgtg gtgatgttga agaaaaccct ggtccaatgt ctagactgga caagagcaaa
     5821 gtcataaacg gagctctgga attactcaat ggtgtcggta tcgaaggcct gacgacaagg
     5881 aaactcgctc aaaagctggg agttgagcag cctaccctgt actggcacgt gaagaacaag
     5941 cgggccctgc tcgatgccct gccaatcgag atgctggaca ggcatcatac ccacttctgc
     6001 cccctggaag gcgagtcatg gcaagacttt ctgcggaaca acgccaagtc ataccgctgt
     6061 gctctcctct cacatcgcga cggggctaaa gtgcatctcg gcacccgccc aacagagaaa
     6121 cagtacgaaa ccctggaaaa tcagctcgcg ttcctgtgtc agcaaggctt ctccctggag
     6181 aacgcactgt acgctctgtc cgccgtgggc cactttacac tgggctgcgt attggaggaa
     6241 caggagcatc aagtagcaaa agaggaaaga gagacaccta ccaccgattc tatgccccca
     6301 cttctgagac aagcaattga gctgttcgac cggcagggag ccgaacctgc cttccttttc
     6361 ggcctggaac taatcatatg tggcctggag aaacagctaa agtgcgaaag cggcgggccg
     6421 accgacgccc ttgacgattt tgacttagac atgctcccag ccgatgccct tgacgacttt
     6481 gaccttgata tgctgcctgc tgacgctctt gacgattttg accttgacat gctccccggg
     6541 taactaagta aggatcgatc caagatatcg tattcttaac tatgttgctc cttttacgct
     6601 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     6661 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     6721 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     6781 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     6841 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     6901 caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc
     6961 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     7021 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     7081 tcagacgagt cggatctccc tttgggccgc ctccccgcct gtttcgcctc ggcgtccgga
     7141 ctagaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     7201 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt
     7261 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     7321 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     7381 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     7441 atctctagca gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     7501 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     7561 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     7621 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     7681 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     7741 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     7801 gaggcttttt tggaggcctt tcaaatatgt atccgctcat gagacaataa ccctgataaa
     7861 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     7921 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     7981 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     8041 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     8101 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     8161 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     8221 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     8281 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     8341 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     8401 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     8461 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     8521 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     8581 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     8641 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     8701 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     8761 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     8821 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     8881 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     8941 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     9001 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     9061 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     9121 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     9181 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     9241 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     9301 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     9361 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     9421 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     9481 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     9541 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     9601 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     9661 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     9721 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     9781 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     9841 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     9901 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
     9961 tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac
    10021 gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga
    10081 agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg
    10141 acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat
    10201 aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
    10261 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
    10321 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
    10381 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
    10441 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
    10501 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
    10561 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
    10621 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
    10681 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
    10741 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
    10801 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
    10861 gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg
    10921 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
    10981 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
    11041 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
    11101 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
    11161 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
    11221 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
    11281 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
    11341 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
    11401 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
    11461 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
    11521 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
    11581 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
    11641 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggac
//