lentiCRISPR v2-Blast

LOCUS       lentiCRISPR v2-Blast           14684 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Mammalian expression of Cas9 and sgRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    lentiCRISPR v2-Blast
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14684)
  AUTHORS   Habashy et al
  TITLE     CRISPR/Cas9-based system for loss-of-function screening
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 14684)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..14684
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(84..104)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        89..418
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      269..404
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     331..350
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        466..513
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             532..906
                     /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    1036..1157
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1073..1092)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1127..1146
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1206..1222)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1206..1222)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1219..1241)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1230..1246
                     /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(1254..1284)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1299..1320
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1437..1454)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1608..2196)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1688..1707)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2367..3227)
                     /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     2990..3009
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3228..3332)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3407..3426)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3598..3977
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3979..4177
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4132..4152
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4195..4375
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4422..4547
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5040..5273
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5458..5502
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    5800..5917
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5968..6208
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     5968..5988
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6139..6158
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8098..8173
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8235..8446
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      8465..8474
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             8471..12574
                     /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             12575..12622
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12623..12646
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             12656..12712
                     /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             12713..13111
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    13136..13724
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(13189..13209)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(13607..13618)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             13796..14029
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(14055..14072)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    14061..14285
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      14331..75
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(14418..14437)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     14628..14649
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
       61 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
      121 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
      181 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
      241 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
      301 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
      361 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
      421 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
      481 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag
      541 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
      601 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
      661 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
      721 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
      781 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
      841 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
      901 gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
      961 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     1021 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     1081 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     1141 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     1201 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     1261 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     1321 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     1381 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     1441 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1501 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1561 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     1621 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1681 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1741 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1801 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1861 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1921 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1981 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2041 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2101 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2161 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2221 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2281 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2341 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2401 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2461 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     2521 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     2581 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     2641 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     2701 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2761 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2821 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2881 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2941 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3001 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3061 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3121 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3181 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3241 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3301 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3361 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     3421 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     3481 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     3541 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     3601 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     3661 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     3721 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     3781 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     3841 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     3901 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     3961 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     4021 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     4081 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     4141 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
     4201 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     4261 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     4321 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     4381 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     4441 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     4501 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     4561 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     4621 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     4681 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     4741 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     4801 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     4861 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     4921 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     4981 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     5041 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     5101 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     5161 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     5221 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     5281 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     5341 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     5401 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     5461 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     5521 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     5581 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     5641 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     5701 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     5761 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     5821 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     5881 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     5941 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     6001 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     6061 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     6121 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     6181 ttggctttat atatcttgtg gaaaggacga aacaccggag acggttgtaa atgagcacac
     6241 aaaatacaca tgctaaaata ttatattcta tgacctttat aaaatcaacc aaaatcttct
     6301 ttttaataac tttagtatca ataattagaa tttttatgtt cctttttgca aacttttaat
     6361 aaaaatgagc aaaataaaaa aacgctagtt ttagtaactc gcgttgtttt cttcaccttt
     6421 aataatagct actccaccac ttgttcctaa gcggtcagct cctgcttcaa tcattttttg
     6481 agcatcttca aatgttctaa ctccaccagc tgctttaact aaagcattgt ctttaacaac
     6541 tgacttcatt agtttaacat cttcaaatgt tgcacctgat tttgaaaatc ctgttgatgt
     6601 tttaacaaat tctaatccag cttcaacagc tatttcacaa gctttcatga tttcttcttt
     6661 tgttaataaa caattttcca taatacattt aacaacatgt gatccagctg ctttttttac
     6721 agctttcatg tcttctaaaa ctaattcata atttttgtct tttaatgcac caatatttaa
     6781 taccatatca atttctgttg caccatcttt aattgcttca gaaacttcga atgcttttgt
     6841 agctgttgtg catgcaccta gaggaaaacc tacaacattt gttattccta catttgtgcc
     6901 ttttaataat tctttacaat agcttgttca atatgaatta acacaaactg ttgcaaaatc
     6961 aaattcaatt gcttcatcac ataattgttt aatttcagct ttcgtagcat cttgttttaa
     7021 taatgtgtga tctatatatt tgtttagttt cattttttct cctatatatt catttttaat
     7081 tttaattctt taataatttc gtctacttta actttagcgt tttgaacaga ttcaccaaca
     7141 cctataaaat aaatttttag tttaggttca gttccacttg ggcgaacagc aaatcatgac
     7201 ttatcttcta aataaaattt tagtaagtct tgtcctggca tattatacat tccatcgatg
     7261 tagtcttcaa cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat
     7321 gatttcatta atggttcaat ttttaatttc ttttcttctg gtttaaaatt caagtttaaa
     7381 gtgaaagtgt aatatgcacc catttcttta aataaatctt ctaaatagtc tactaatgtt
     7441 ttattttgtt ttttataaaa tcaagcagcc tctgctatta atatagaagc ttgtattcca
     7501 tctttatctc tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca
     7561 aaatttaatc cgttatcttc ttctttagca atttctctac ccattcattt aaatccagtt
     7621 aaagttttta caatattaac tccatatttt tcatgagcga ttctatcacc caaatcactt
     7681 gttacaaaac ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt
     7741 gataattttc aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga
     7801 ccatcatgtt ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata
     7861 tctgcatcat gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga
     7921 tttggattta caacattttt aaatgtttca tcttcaaatg catgctcttc aacctcaata
     7981 acgttatatc ctgattcacg taatattttt ggggtaaatt tagttcctgt tccattaact
     8041 gcgctaaaaa taatttttaa atctttttta gcttcttgct cttttttgta cgtctctgtt
     8101 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     8161 accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc
     8221 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     8281 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     8341 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     8401 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag
     8461 cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg
     8521 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     8581 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     8641 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     8701 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     8761 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     8821 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     8881 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     8941 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     9001 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     9061 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     9121 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     9181 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     9241 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     9301 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     9361 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     9421 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     9481 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     9541 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     9601 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     9661 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     9721 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     9781 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     9841 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     9901 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     9961 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
    10021 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
    10081 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
    10141 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
    10201 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
    10261 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
    10321 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
    10381 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
    10441 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
    10501 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
    10561 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
    10621 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
    10681 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
    10741 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
    10801 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
    10861 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
    10921 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
    10981 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
    11041 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
    11101 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
    11161 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
    11221 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
    11281 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
    11341 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
    11401 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
    11461 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
    11521 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
    11581 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
    11641 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
    11701 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
    11761 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
    11821 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
    11881 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
    11941 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
    12001 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
    12061 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
    12121 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
    12181 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
    12241 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
    12301 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
    12361 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
    12421 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
    12481 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
    12541 gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc
    12601 tggacaggct aagaagaaga aagattacaa agacgatgac gataagggat ccggcgcaac
    12661 aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cgatggccaa
    12721 gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat
    12781 ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt
    12841 cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg
    12901 cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa
    12961 caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg
    13021 gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt
    13081 gctgccctct ggttatgtgt gggagggcta agaattcgat atcaagctta tcggtaatca
    13141 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
    13201 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
    13261 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
    13321 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
    13381 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
    13441 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
    13501 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
    13561 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
    13621 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
    13681 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca
    13741 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
    13801 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
    13861 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
    13921 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
    13981 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag ggcccgttta
    14041 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
    14101 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
    14161 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
    14221 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
    14281 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
    14341 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
    14401 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
    14461 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
    14521 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
    14581 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
    14641 ccaaactgga acaacactca accctatctc ggtctattct tttg
//