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
//
Caution:
Product is for research use only!
