lentiCRISPR v2

LOCUS       lentiCRISPR v2               14873 bp ds-DNA     circular SYN 10-JAN-2018
DEFINITION  Replaces original lentiCRISPRv1 (Addgene Plasmid 49535) and produces
            ~10-fold higher titer virus. 3rd generation lentiviral backbone..
ACCESSION   .
VERSION     .
KEYWORDS    lentiCRISPR v2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14873)
  AUTHORS   Sanjana NE, Shalem O, Zhang F
  TITLE     Improved vectors and genome-wide libraries for CRISPR screening.
  JOURNAL   Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.
  PUBMED    25075903
COMMENT     This file is created by Vector NTI
            http://www.biofeng.com/
COMMENT     ORIGDB|GenBank
COMMENT     VNTAUTHORNAME|biofeng.com|
COMMENT     VNTNAME|lentiCRISPR v2|
FEATURES             Location/Qualifiers
     source          1..14873
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1062..1295
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1990..2230
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1990..2010
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2161..2180
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4120..4195
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4257..4468
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4487..4496
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4493..8596
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             8597..8644
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8645..8668
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             8678..8734
                     /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             8735..9331
                     /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     9228..9248
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9347..9935
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9400..9420)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9818..9829)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10007..10240
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10266..10283)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10272..10496
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10542..10970
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10629..10648)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10839..10860
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10979..10999)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10984..11313
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11164..11299
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11226..11245
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11361..11408
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11427..11801
                     /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    11931..12052
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11968..11987)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12022..12041
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12101..12117)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12101..12117)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12114..12136)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12125..12141
                     /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(12149..12179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12194..12215
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12332..12349)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12503..13091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12583..12602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(13262..14122)
                     /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     13885..13904
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14123..14227)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(14302..14321)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        14493..14872
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
      961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
     1981 taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2041 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2101 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2161 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     2221 tggaaaggac gaaacaccgg agacggttgt aaatgagcac acaaaataca catgctaaaa
     2281 tattatattc tatgaccttt ataaaatcaa ccaaaatctt ctttttaata actttagtat
     2341 caataattag aatttttatg ttcctttttg caaactttta ataaaaatga gcaaaataaa
     2401 aaaacgctag ttttagtaac tcgcgttgtt ttcttcacct ttaataatag ctactccacc
     2461 acttgttcct aagcggtcag ctcctgcttc aatcattttt tgagcatctt caaatgttct
     2521 aactccacca gctgctttaa ctaaagcatt gtctttaaca actgacttca ttagtttaac
     2581 atcttcaaat gttgcacctg attttgaaaa tcctgttgat gttttaacaa attctaatcc
     2641 agcttcaaca gctatttcac aagctttcat gatttcttct tttgttaata aacaattttc
     2701 cataatacat ttaacaacat gtgatccagc tgcttttttt acagctttca tgtcttctaa
     2761 aactaattca taatttttgt cttttaatgc accaatattt aataccatat caatttctgt
     2821 tgcaccatct ttaattgctt cagaaacttc gaatgctttt gtagctgttg tgcatgcacc
     2881 tagaggaaaa cctacaacat ttgttattcc tacatttgtg ccttttaata attctttaca
     2941 atagcttgtt caatatgaat taacacaaac tgttgcaaaa tcaaattcaa ttgcttcatc
     3001 acataattgt ttaatttcag ctttcgtagc atcttgtttt aataatgtgt gatctatata
     3061 tttgtttagt ttcatttttt ctcctatata ttcattttta attttaattc tttaataatt
     3121 tcgtctactt taactttagc gttttgaaca gattcaccaa cacctataaa ataaattttt
     3181 agtttaggtt cagttccact tgggcgaaca gcaaatcatg acttatcttc taaataaaat
     3241 tttagtaagt cttgtcctgg catattatac attccatcga tgtagtcttc aacattaaca
     3301 actttaagtc cagcaatttg agttaagggt gttgctctca atgatttcat taatggttca
     3361 atttttaatt tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt gtaatatgca
     3421 cccatttctt taaataaatc ttctaaatag tctactaatg ttttattttg ttttttataa
     3481 aatcaagcag cctctgctat taatatagaa gcttgtattc catctttatc tctagctgag
     3541 tcatcaatta catatccata actttcttca taagcaaaaa caaaatttaa tccgttatct
     3601 tcttctttag caatttctct acccattcat ttaaatccag ttaaagtttt tacaatatta
     3661 actccatatt tttcatgagc gattctatca cccaaatcac ttgttacaaa acttgaatat
     3721 agagccggat tttttggaat gctatttaag cgttttagat ttgataattt tcaatcaatt
     3781 aaaattggtc ctgtttgatt tccatctaat cttacaaaat gaccatcatg ttttattgcc
     3841 attccaaatc tgtcagcatc tgggtcattc ataataataa tatctgcatc atgtttaata
     3901 ccatattcaa gcggtatttt tcatgcagga tcaaattctg gatttggatt tacaacattt
     3961 ttaaatgttt catcttcaaa tgcatgctct tcaacctcaa taacgttata tcctgattca
     4021 cgtaatattt ttggggtaaa tttagttcct gttccattaa ctgcgctaaa aataattttt
     4081 aaatcttttt tagcttcttg ctcttttttg tacgtctctg ttttagagct agaaatagca
     4141 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     4201 tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
     4261 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg
     4321 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     4381 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     4441 tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa
     4501 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
     4561 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     4621 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     4681 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     4741 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     4801 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     4861 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     4921 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     4981 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     5041 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     5101 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     5161 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     5221 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     5281 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     5341 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     5401 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     5461 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     5521 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     5581 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     5641 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     5701 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     5761 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     5821 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     5881 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     5941 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     6001 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     6061 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     6121 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     6181 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     6241 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     6301 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     6361 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     6421 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     6481 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     6541 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     6601 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     6661 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     6721 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     6781 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     6841 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     6901 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     6961 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     7021 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     7081 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     7141 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     7201 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     7261 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     7321 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     7381 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     7441 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     7501 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     7561 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     7621 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     7681 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     7741 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     7801 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     7861 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     7921 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     7981 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     8041 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     8101 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     8161 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     8221 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     8281 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     8341 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     8401 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     8461 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     8521 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     8581 tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa
     8641 gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa
     8701 acaagccgga gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct
     8761 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     8821 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     8881 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     8941 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     9001 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     9061 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt
     9121 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     9181 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     9241 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     9301 ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga
     9361 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     9421 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     9481 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     9541 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     9601 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     9661 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     9721 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     9781 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     9841 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     9901 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
     9961 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
    10021 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
    10081 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
    10141 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
    10201 tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg
    10261 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
    10321 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
    10381 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
    10441 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
    10501 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
    10561 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
    10621 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
    10681 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
    10741 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
    10801 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
    10861 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
    10921 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
    10981 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
    11041 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
    11101 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
    11161 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
    11221 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
    11281 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
    11341 ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat
    11401 aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca
    11461 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
    11521 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
    11581 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
    11641 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
    11701 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
    11761 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg
    11821 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
    11881 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
    11941 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
    12001 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
    12061 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
    12121 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
    12181 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
    12241 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
    12301 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
    12361 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
    12421 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
    12481 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
    12541 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
    12601 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
    12661 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
    12721 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
    12781 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
    12841 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
    12901 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
    12961 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
    13021 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
    13081 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
    13141 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
    13201 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
    13261 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
    13321 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
    13381 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
    13441 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
    13501 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
    13561 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
    13621 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
    13681 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
    13741 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
    13801 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
    13861 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
    13921 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
    13981 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
    14041 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
    14101 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
    14161 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
    14221 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
    14281 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
    14341 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
    14401 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
    14461 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
    14521 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
    14581 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
    14641 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
    14701 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
    14761 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
    14821 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgg
//