LOCUS       sequence-147481-       14849 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Introduce sgRNA into a lentiviral vector (LentiCRISPR V2) which 
            contains eSpCas9 and puromycin cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14849)
  AUTHORS   Sikder RK, Ellithi M, Uzzo RN, Weader D, Metz AL, Behbahani A, 
            McKenzie ER, El-Deiry WS, Abbosh PH
  TITLE     Differential effects of clinically-relevant N- vs C-terminal 
            truncating CDKN1A mutations on cisplatin sensitivity in bladder 
            cancer.
  JOURNAL   Mol Cancer Res. 2020 Dec 3. pii: 1541-7786.MCR-19-1200. doi: 
            10.1158/1541-7786.MCR-19-1200.
  PUBMED    33272936
REFERENCE   2  (bases 1 to 14849)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cancer 
            Res. 2020 Dec 3. pii: 1541-7786.MCR-19-1200. doi: 
            10.1158/1541-7786.MCR-19-1200."
COMMENT     Sequence Label: pLentiCRISPR-E
FEATURES             Location/Qualifiers
     source          1..14849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2608..2856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2608..2628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2779..2798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4738..4813
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4875..5086
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5105..5114
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5111..7651,7652..7654,7655..8116,8117..8119,8120..8287,
                     8288..8290,8291..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)"
                     /label=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"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9272..9328
                     /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             9329..9925
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     9822..9842
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9941..10529
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9994..10014)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10412..10423)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10601..10834
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10860..10877)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10866..11090
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11136..11564
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11223..11242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11433..11454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11573..11593)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11578..11907
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11758..11893
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11820..11839
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11955..12002
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12021..12395
                     /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    12525..12646
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12562..12581)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12616..12635
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12695..12711)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12695..12711)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12708..12730)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12719..12735
                     /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(join(12743..12749,12750..12767,12768..12773))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12788..12809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12926..12943)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13097..13685)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13177..13196)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13856..14647,14648..14716))
                     /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     14479..14498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14717..14821)
                     /gene="bla"
                     /label=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
//
