pL-CRISPR.EFS.tRFP

LOCUS       pL-CRISPR.EFS.tRFP               11701 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Lentiviral CRISPR-Cas9 delivery for SpCas9 and sgRNA. Coexpresses 
            tagRFP via P2A cleavage site. EFS Promoter driven.
ACCESSION   .
VERSION     .
KEYWORDS    pL-CRISPR.EFS.tRFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11701)
  AUTHORS   Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey 
            ME, Thielke A, Aster JC, Regev A, Ebert BL
  TITLE     Generation of mouse models of myeloid malignancy with combinatorial 
            genetic lesions using CRISPR-Cas9 genome editing.
  JOURNAL   Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951.
  PUBMED    24952903
REFERENCE   2  (bases 1 to 11701)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..11701
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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"
     promoter        1717..1957
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1984..2059
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2107..2224
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2293..2504
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2524..2533
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2533..2556
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2563..2583
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2608..6708
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6709..6756
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6772..6828
                     /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             6829..7542
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    7558..8146
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7611..7631)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8029..8040)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8218..8451
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8529..8650
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8566..8585)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8620..8639
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8690..8825
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     8752..8771
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(8845..8864)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8846..8864)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8874..8891)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8874..8890)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8883..8905)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9032..9487
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9119..9138)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9329..9350
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9513..9617
                     /gene="bla"
                     /label=AmpR promoter
     CDS             9618..10478
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(9836..9855)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10649..11237
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11138..11157
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11391..11408
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11525..11546
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        11561..11591
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11599..11615
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     11604..11626
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11623..11639
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11623..11639
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11658..11678
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11660..11678
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cgtgaccgtc
     1981 tctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     2041 agtggcaccg agtcggtgct tttttgaatt ctagatcttg agacaaatgg cagtattcat
     2101 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     2161 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     2221 ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg agtggctccg
     2281 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
     2341 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
     2401 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
     2461 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga
     2521 tccgccacca tggattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag
     2581 gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct ggacatcggc
     2641 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     2701 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     2761 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     2821 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     2881 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     2941 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     3001 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     3061 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     3121 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     3181 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     3241 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     3301 aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc
     3361 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     3421 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     3481 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     3541 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     3601 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     3661 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     3721 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     3781 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     3841 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     3901 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     3961 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     4021 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
     4081 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     4141 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
     4201 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
     4261 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     4321 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     4381 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     4441 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     4501 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     4561 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     4621 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     4681 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     4741 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     4801 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     4861 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     4921 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     4981 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     5041 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     5101 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
     5161 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
     5221 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
     5281 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     5341 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     5401 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
     5461 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
     5521 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
     5581 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
     5641 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
     5701 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
     5761 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
     5821 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
     5881 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
     5941 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
     6001 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
     6061 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
     6121 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
     6181 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
     6241 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
     6301 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
     6361 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
     6421 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
     6481 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
     6541 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
     6601 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
     6661 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgtcctgct
     6721 gctactaaga aagctggtca agctaagaaa aagaaagcta gcggcagcgg cgccaccaac
     6781 ttcagcctgc tgaagcaggc cggcgacgtg gaggagaacc ccggccccat ggtgtctaag
     6841 ggcgaagagc tgattaagga gaacatgcac atgaagctgt atatggaggg caccgtgaac
     6901 aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
     6961 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctacc
     7021 agcttcatgt acggcagcag aaccttcatc aaccacaccc agggcatccc cgacttcttt
     7081 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     7141 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
     7201 atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac actcggctgg
     7261 gaggccaaca ccgagatgct gtaccccgct gacggcggcc tggaaggcag aagcgacatg
     7321 gccctgaagc tcgtgggcgg gggccacctg atctgcaact tcaagaccac atacagatcc
     7381 aagaaacccg ctaagaacct caagatgccc ggcgtctact atgtggacca cagactggaa
     7441 agaatcaagg aggccgacaa agagacctac gtcgagcagc acgaggtggc tgtggccaga
     7501 tactgcgacc tccctagcaa actggggcac aaacttaatt gaacgcgtta agtcgacaat
     7561 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     7621 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     7681 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     7741 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     7801 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     7861 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     7921 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     7981 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     8041 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     8101 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac
     8161 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     8221 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     8281 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     8341 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     8401 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata
     8461 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     8521 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     8581 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     8641 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
     8701 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     8761 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     8821 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     8881 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     8941 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     9001 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     9061 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     9121 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     9181 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     9241 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     9301 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     9361 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     9421 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     9481 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     9541 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     9601 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     9661 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     9721 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     9781 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     9841 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     9901 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     9961 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
    10021 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
    10081 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
    10141 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
    10201 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
    10261 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
    10321 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
    10381 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
    10441 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
    10501 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
    10561 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
    10621 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
    10681 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
    10741 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
    10801 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
    10861 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
    10921 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
    10981 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
    11041 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
    11101 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
    11161 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
    11221 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
    11281 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
    11341 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
    11401 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
    11461 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
    11521 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
    11581 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
    11641 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag
    11701 c
//