pLentiCRISPR-E

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  • Model: PVT10914
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pLentiCRISPR-E

Catalog No. PVT10914
Packing 2ug

 

pLentiCRISPR-E Information

Function Mammal Editing plasmids

Plasmid classification: gene editing vector; mammalian cell CRISPR-CAS9-sgRNA plasmid

Prokaryotic resistance: ampicillin Amp

Screening markers: purinomycin Puro

Cloned strain: Escherichia coli Stbl3

Culture conditions: LB medium, 37 C

Note: high copy

 

pLentiCRISPR-E Description

(Empty Backbone) Introduce sgRNA into a lentiviral vector (LentiCRISPR V2) which contains eSpCas9 and puromycin cassette
 
This plasmid is derived from the Feng Zhang lab’s pLentiCRISPR V2 plasmid (the one that has a highly active puromycin variant, Addgene #52961), and the eSpCas9(1.1) plasmid (Addgene #71814). 
 
Cloning method:
A Fragment of eSpCas9 was amplified from #71814 using primers:

#2eSpCas9-F-EcoRV TTCTGGAAGATATCGTGCTGACCCTGACAC
#2eSpCas9-R-BamHI aaCGGtGGATCCCTTTTTCTTTTTTGCCTGGCCGGC

 
The reverse primer introduces a BamHI site which is present in #52961 but not #71814.  Primers amplify the 3’ half of eSpCas9 which contains all 3 point mutations described in Slaymaker et al (Science 351:84).
Plasmid #52961 and the PCR product were digested with BamHI and EcoRV. #52961 was also exposed to phosphatase. Fragments were ligated, screened for insert, and sequenced.
 
How to use:
You can clone sgRNAs into the vector just like you would using pLentiCRISPR V2 using the enzyme BsmBI as previously described by the Zhang lab. This will liberate a 2.2kb fragment on agarose gel. The larger fragment can be gel-purified to use as backbone, or the circular plasmid can be used in Golden Gate reactions.
 
Notes:
eSpCas9 is reportedly very sensitive to mismatches, including mismatches created by adding a 5’G to increase expression from a U6 promoter (which drives sgRNA expression in pLentiCRISPR-E). Also note that the Kleinsteiver et al Cas9-HF1 construct (Nature 529:490) loses a significant amount of efficiency when using truncated guides, and this may be true for eSpCas9 also but hasn’t been formally tested at the time of plasmid submission.  For this reason, consider using guides that start with a native 5’G.

 

pLentiCRISPR-E Sequence

LOCUS       Exported               14849 bp ds-DNA     circular SYN 04-NOV-2016

FEATURES             Location/Qualifiers

     source          1..14849

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     enhancer        238..617

                     /note="CMV enhancer"

                     /note="human cytomegalovirus immediate early enhancer"

     promoter        619..817

                     /note="CMV promoter"

                     /note="human cytomegalovirus (CMV) immediate early 

                     promoter"

     LTR             835..1015

                     /note="5' LTR (truncated)"

                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"

     misc_feature    1062..1187

                     /note="HIV-1 Psi"

                     /note="packaging signal of human immunodeficiency virus 

                     type 1"

     misc_feature    1680..1913

                     /note="RRE"

                     /note="The Rev response element (RRE) of HIV-1 allows for 

                     Rev-dependent mRNA export from the nucleus to the 

                     cytoplasm."

     misc_feature    2440..2557

                     /note="cPPT/CTS"

                     /note="central polypurine tract and central termination 

                     sequence of HIV-1"

     promoter        2608..2848

                     /note="U6 promoter"

                     /note="RNA polymerase III promoter for human U6 snRNA"

     misc_RNA        4738..4813

                     /note="gRNA scaffold"

                     /note="guide RNA scaffold for the Streptococcus pyogenes 

                     CRISPR/Cas9 system"

     promoter        4875..5086

                     /note="EF-1-alpha core promoter"

                     /note="core promoter for human elongation factor 

                     EF-1-alpha"

     CDS             5111..9214

                     /codon_start=1

                     /product="Cas9 endonuclease from the Streptococcus pyogenes

                     Type II CRISPR/Cas system, mutated to improve targeting 

                     specificity (Slaymaker et al., 2016)"

                     /note="eSpCas9(1.1)"

                     /note="carries the mutations K848A, K1003A, and R1060A"

                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK

                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES

                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK

                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL

                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ

                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR

                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL

                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG

                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY

                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD

                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER

                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM

                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR

                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY

                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVP

                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH

                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL

                     NAVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT

                     EITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK

                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI

                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE

                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD

                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL

                     DATLIHQSITGLYETRIDLSQLGGD"

     CDS             9215..9262

                     /codon_start=1

                     /product="bipartite nuclear localization signal from 

                     nucleoplasmin"

                     /note="nucleoplasmin NLS"

                     /translation="KRPAATKKAGQAKKKK"

     CDS             9272..9328

                     /codon_start=1

                     /product="2A peptide from porcine teschovirus-1 

                     polyprotein"

                     /note="P2A"

                     /note="Eukaryotic ribosomes fail to insert a peptide bond 

                     between the Gly and Pro residues, yielding separate 

                     polypeptides."

                     /translation="ATNFSLLKQAGDVEENPGP"

     CDS             9329..9925

                     /codon_start=1

                     /gene="pac from Streptomyces alboniger"

                     /product="puromycin N-acetyltransferase"

                     /note="PuroR"

                     /note="confers resistance to puromycin"

                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV

                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA

                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA

                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"

     misc_feature    9941..10529

                     /note="WPRE"

                     /note="woodchuck hepatitis virus posttranscriptional 

                     regulatory element"

     CDS             complement(10412..10423)

                     /codon_start=1

                     /product="Factor Xa recognition and cleavage site"

                     /note="Factor Xa site"

                     /translation="IEGR"

     LTR             10601..10834

                     /note="3' LTR (Delta-U3)"

                     /note="self-inactivating 3' long terminal repeat (LTR) from

                     HIV-1"

     polyA_signal    10866..11090

                     /note="bGH poly(A) signal"

                     /note="bovine growth hormone polyadenylation signal"

     rep_origin      11136..11564

                     /direction=RIGHT

                     /note="f1 ori"

                     /note="f1 bacteriophage origin of replication; arrow 

                     indicates direction of (+) strand synthesis"

     promoter        11578..11907

                     /note="SV40 promoter"

                     /note="SV40 enhancer and early promoter"

     rep_origin      11758..11893

                     /note="SV40 ori"

                     /note="SV40 origin of replication"

     promoter        11955..12002

                     /note="EM7 promoter"

                     /note="synthetic bacterial promoter "

     CDS             12021..12395

                     /codon_start=1

                     /gene="Sh ble from Streptoalloteichus hindustanus"

                     /product="antibiotic-binding protein"

                     /note="BleoR"

                     /note="confers resistance to bleomycin, phleomycin, and 

                     Zeocin(TM)"

                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD

                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR

                     EFALRDPAGNCVHFVAEEQD"

     polyA_signal    12525..12646

                     /note="SV40 poly(A) signal"

                     /note="SV40 polyadenylation signal"

     primer_bind     complement(12695..12711)

                     /note="M13 rev"

                     /note="common sequencing primer, one of multiple similar 

                     variants"

     protein_bind    12719..12735

                     /bound_moiety="lac repressor encoded by lacI"

                     /note="lac operator"

                     /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(12743..12773)

                     /note="lac promoter"

                     /note="promoter for the E. coli lac operon"

     protein_bind    12788..12809

                     /bound_moiety="E. coli catabolite activator protein"

                     /note="CAP binding site"

                     /note="CAP binding activates transcription in the presence 

                     of cAMP."

     rep_origin      complement(13097..13685)

                     /direction=LEFT

                     /note="ori"

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 

                     replication"

     CDS             complement(13856..14716)

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /note="AmpR"

                     /note="confers resistance to ampicillin, carbenicillin, and

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     promoter        complement(14717..14821)

                     /gene="bla"

                     /note="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 ggttctagag

     5101 cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg

     5161 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa

     5221 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga

     5281 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa

     5341 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt

     5401 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc

     5461 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca

     5521 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc

     5581 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga

     5641 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga

     5701 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag

     5761 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct

     5821 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga

     5881 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa

     5941 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc

     6001 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct

     6061 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc

     6121 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa

     6181 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa

     6241 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga

     6301 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg

     6361 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg

     6421 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag

     6481 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa

     6541 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa

     6601 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta

     6661 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc

     6721 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg

     6781 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc

     6841 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct

     6901 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga

     6961 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa

     7021 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac

     7081 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa

     7141 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat

     7201 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg

     7261 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat

     7321 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga

     7381 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag

     7441 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa

     7501 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca

     7561 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga

     7621 tgtggaccat atcgtgcctc agagctttct ggccgacgac tccatcgaca acaaggtgct

     7681 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa

     7741 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt

     7801 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat

     7861 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc

     7921 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac

     7981 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga

     8041 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct

     8101 gatcaaaaag taccctgcgc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga

     8161 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt

     8221 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat

     8281 ccggaaggcg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg

     8341 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa

     8401 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga

     8461 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc

     8521 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact

     8581 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa

     8641 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa

     8701 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc

     8761 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta

     8821 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct

     8881 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc

     8941 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca

     9001 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa

     9061 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac

     9121 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga

     9181 gacacggatc gacctgtctc agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc

     9241 cggccaggca aaaaagaaaa agggatccgg cgcaacaaac ttctctctgc tgaaacaagc

     9301 cggagatgtc gaagagaatc ctggaccgac cgagtacaag cccacggtgc gcctcgccac

     9361 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc

     9421 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact

     9481 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc

     9541 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg

     9601 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct

     9661 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc

     9721 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga

     9781 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga

     9841 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg

     9901 catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa

     9961 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata

    10021 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc

    10081 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg

    10141 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac

    10201 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat

    10261 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt

    10321 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat

    10381 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc

    10441 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag

    10501 tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca

    10561 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc

    10621 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga

    10681 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct

    10741 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc

    10801 agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc gctgatcagc

    10861 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt

    10921 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca

    10981 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga

    11041 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc

    11101 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag

    11161 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc

    11221 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc

    11281 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa

    11341 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg

    11401 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac

    11461 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta

    11521 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg

    11581 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg

    11641 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt

    11701 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc

    11761 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt

    11821 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc

    11881 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga

    11941 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg

    12001 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc

    12061 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg

    12121 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc

    12181 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc

    12241 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca

    12301 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca

    12361 actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca

    12421 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga

    12481 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag

    12541 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt

    12601 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac

    12661 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt

    12721 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg

    12781 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt

    12841 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt

    12901 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc

    12961 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg

    13021 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg

    13081 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac

    13141 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg

    13201 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct

    13261 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg

    13321 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct

    13381 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac

    13441 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt

    13501 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc

    13561 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca

    13621 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat

    13681 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac

    13741 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt

    13801 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc

    13861 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg

    13921 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg

    13981 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc

    14041 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta

    14101 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg

    14161 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct

    14221 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta

    14281 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg

    14341 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga

    14401 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt

    14461 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca

    14521 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt

    14581 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt

    14641 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga

    14701 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt

    14761 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc

    14821 gcacatttcc ccgaaaagtg ccacctgac

//

 

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