LOCUS       pRC0494_Lenti-CRISPR-V2_Cas9-T2A-eGFP-P2A-Puro       15650 bp ds-DNA     circular SYN 24-DEC-2020
DEFINITION  Replaces original lentiCRISPRv1 (Addgene Plasmid 49535) and
            produces ~10-fold higher titer virus. 3rd generation lentiviral
            backbone.
ACCESSION
VERSION     .
KEYWORDS    lentiCRISPR v2.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 14873)
  AUTHORS   Sanjana, NE, Shalem, O, Zhang and F
  TITLE     Improved vectors and genome-wide libraries for CRISPR screening.
  JOURNAL   Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.
  PUBMED    25075903
REFERENCE   2  (bases 1 to 14873)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 10, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..15650
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..199
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /label="CMV promoter"
     primer_bind     154..174
                     /note="Human CMV immediate early promoter, forward primer"
                     /label="CMV-F"
     LTR             217..397
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /label="5' LTR (truncated)"
     misc_feature    444..569
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
                     /label="HIV-1 Psi"
     misc_feature    1062..1295
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to
                     thecytoplasm."
                     /label="RRE"
     misc_feature    1822..1939
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
                     /label="cPPT/CTS"
     promoter        1990..2230
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /label="U6 promoter"
     primer_bind     1990..2010
                     /note="Human U6 promoter, forward primer"
                     /label="hU6-F"
     primer_bind     2161..2180
                     /note="Human U6 promoter, forward primer"
                     /label="LKO.1 5'"
     PCR_primer      2217..2239
                     /created_by="charir2"
                     /label="Primer_592"
     source          <2240..>4119
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_RNA        4120..4195
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /label="gRNA scaffold"
     promoter        4257..4468
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
                     /label="EF-1-alpha core promoter"
     PCR_primer      complement(4307..4326)
                     /created_by="charir2"
                     /label="Primer_667"
     regulatory      4487..4496
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation
                      of translation (Kozak, 1987)"
     CDS             4493..8596
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK
                     KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE
                     ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH
                     MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK
                     SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD
                     NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
                     LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK
                     LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
                     YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL
                     PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE
                     DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT
                     LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF
                     LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT
                     VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE
                     HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV
                     LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA
                     GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG
                     KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
                     MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
                     AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF
                     ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH
                     KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
                     PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /label="Cas9"
     PCR_primer      8068..8087
                     /created_by="charir2"
                     /label=528
     PCR_primer      complement(8212..8232)
                     /created_by="charir2"
                     /label=1907
     CDS             8597..8644
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /translation="KRPAATKKAGQAKKKK"
                     /label="nucleoplasmin NLS"
     CDS             8645..8668
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase
                      cleavage site"
                     /translation="DYKDDDDK"
                     /label="FLAG"
     source          <8669..>8677
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    8669..8677
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     source          <8678..>9445
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             8678..8731
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding
                     separatepolypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
                     /label="T2A"
     PCR_primer      complement(8712..8731)
                     /created_by="charir2"
                     /label="T2A-R"
     CDS             8732..9445
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG
                     IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHM
                     VLLEFVTAAGITLGMDELYK"
                     /label="EGFP"
     primer_bind     complement(8774..8795)
                     /note="EGFP, reverse primer"
                     /label="EGFP-N"
     primer_bind     complement(9035..9054)
                     /note="For distinguishing EGFP variants, reverse primer"
                     /label="EXFP-R"
     primer_bind     9382..9403
                     /note="EGFP, forward primer"
                     /label="EGFP-C"
     PCR_primer      complement(9426..9445)
                     /created_by="charir2"
                     /label="GFP-R"
     misc_feature    9446..9454
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     CDS             9455..9511
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding
                     separatepolypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
                     /label="P2A"
     CDS             9512..10108
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLE
                     TSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
                     /label="PuroR"
     PCR_primer      complement(9656..9676)
                     /created_by="charir2"
                     /label=394
     primer_bind     10005..10025
                     /note="Puromycin resistance gene, forward primer"
                     /label="Puro-F"
     misc_feature    10124..10712
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /label="WPRE"
     primer_bind     complement(10177..10197)
                     /note="WPRE, reverse primer"
                     /label="WPRE-R"
     PCR_primer      10222..10242
                     /created_by="charir2"
                     /label="WPRE-F"
     PCR_primer      complement(10336..10355)
                     /created_by="charir2"
                     /label="WPRE-RC-R"
     PCR_primer      complement(10581..10598)
                     /created_by="charir2"
                     /label="Primer_1787"
     CDS             complement(10595..10606)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
                     /label="Factor Xa site"
     LTR             10784..11017
                     /note="self-inactivating 3' long terminal repeat (LTR)
                     from HIV-1"
                     /label="3' LTR (Delta-U3)"
     primer_bind     complement(11043..11060)
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
                     /label="BGH-rev"
     polyA_signal    11049..11273
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      11319..11747
                     /direction="RIGHT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     primer_bind     complement(11406..11425)
                     /note="F1 origin, reverse primer"
                     /label="F1ori-R"
     primer_bind     11616..11637
                     /note="F1 origin, forward primer"
                     /label="F1ori-F"
     primer_bind     complement(11756..11776)
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
                     /label="pBABE 3'"
     promoter        11761..12090
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     rep_origin      11941..12076
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     12003..12022
                     /note="SV40 promoter/origin, forward primer"
                     /label="SV40pro-F"
     promoter        12138..12185
                     /note="synthetic bacterial promoter"
                     /label="EM7 promoter"
     CDS             12204..12578
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRD
                     DVTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPW
                     GREFALRDPAGNCVHFVAEEQD"
                     /label="BleoR"
     polyA_signal    12708..12829
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(12745..12764)
                     /note="SV40 polyA, reverse primer"
                     /label="SV40pA-R"
     primer_bind     12799..12818
                     /note="SV40 polyA terminator, reverse primer"
                     /label="EBV-rev"
     primer_bind     complement(12878..12894)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 rev"
     primer_bind     complement(12878..12894)
                     /note="In lacZ gene. Also called M13-rev"
                     /label="M13 Reverse"
     primer_bind     complement(12891..12913)
                     /note="In lacZ gene"
                     /label="M13/pUC Reverse"
     protein_bind    12902..12918
                     /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)."
                     /label="lac operator"
     promoter        complement(12926..12956)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    12971..12992
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     primer_bind     complement(13109..13126)
                     /note="L4440 vector, forward primer"
                     /label="L4440"
     rep_origin      complement(13280..13868)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     primer_bind     complement(13360..13379)
                     /note="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
     CDS             complement(14039..14899)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /label="AmpR"
     primer_bind     14662..14681
                     /note="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     promoter        complement(14900..15004)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     complement(15079..15098)
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /label="pRS-marker"
     enhancer        15270..15649
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
ORIGIN      
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
      961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
     1981 taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2041 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2101 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2161 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     2221 tggaaaggac gaaacaccgg agacggttgt aaatgagcac acaaaataca catgctaaaa
     2281 tattatattc tatgaccttt ataaaatcaa ccaaaatctt ctttttaata actttagtat
     2341 caataattag aatttttatg ttcctttttg caaactttta ataaaaatga gcaaaataaa
     2401 aaaacgctag ttttagtaac tcgcgttgtt ttcttcacct ttaataatag ctactccacc
     2461 acttgttcct aagcggtcag ctcctgcttc aatcattttt tgagcatctt caaatgttct
     2521 aactccacca gctgctttaa ctaaagcatt gtctttaaca actgacttca ttagtttaac
     2581 atcttcaaat gttgcacctg attttgaaaa tcctgttgat gttttaacaa attctaatcc
     2641 agcttcaaca gctatttcac aagctttcat gatttcttct tttgttaata aacaattttc
     2701 cataatacat ttaacaacat gtgatccagc tgcttttttt acagctttca tgtcttctaa
     2761 aactaattca taatttttgt cttttaatgc accaatattt aataccatat caatttctgt
     2821 tgcaccatct ttaattgctt cagaaacttc gaatgctttt gtagctgttg tgcatgcacc
     2881 tagaggaaaa cctacaacat ttgttattcc tacatttgtg ccttttaata attctttaca
     2941 atagcttgtt caatatgaat taacacaaac tgttgcaaaa tcaaattcaa ttgcttcatc
     3001 acataattgt ttaatttcag ctttcgtagc atcttgtttt aataatgtgt gatctatata
     3061 tttgtttagt ttcatttttt ctcctatata ttcattttta attttaattc tttaataatt
     3121 tcgtctactt taactttagc gttttgaaca gattcaccaa cacctataaa ataaattttt
     3181 agtttaggtt cagttccact tgggcgaaca gcaaatcatg acttatcttc taaataaaat
     3241 tttagtaagt cttgtcctgg catattatac attccatcga tgtagtcttc aacattaaca
     3301 actttaagtc cagcaatttg agttaagggt gttgctctca atgatttcat taatggttca
     3361 atttttaatt tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt gtaatatgca
     3421 cccatttctt taaataaatc ttctaaatag tctactaatg ttttattttg ttttttataa
     3481 aatcaagcag cctctgctat taatatagaa gcttgtattc catctttatc tctagctgag
     3541 tcatcaatta catatccata actttcttca taagcaaaaa caaaatttaa tccgttatct
     3601 tcttctttag caatttctct acccattcat ttaaatccag ttaaagtttt tacaatatta
     3661 actccatatt tttcatgagc gattctatca cccaaatcac ttgttacaaa acttgaatat
     3721 agagccggat tttttggaat gctatttaag cgttttagat ttgataattt tcaatcaatt
     3781 aaaattggtc ctgtttgatt tccatctaat cttacaaaat gaccatcatg ttttattgcc
     3841 attccaaatc tgtcagcatc tgggtcattc ataataataa tatctgcatc atgtttaata
     3901 ccatattcaa gcggtatttt tcatgcagga tcaaattctg gatttggatt tacaacattt
     3961 ttaaatgttt catcttcaaa tgcatgctct tcaacctcaa taacgttata tcctgattca
     4021 cgtaatattt ttggggtaaa tttagttcct gttccattaa ctgcgctaaa aataattttt
     4081 aaatcttttt tagcttcttg ctcttttttg tacgtctctg ttttagagct agaaatagca
     4141 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     4201 tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
     4261 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg
     4321 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     4381 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     4441 tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa
     4501 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
     4561 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     4621 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     4681 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     4741 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     4801 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     4861 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     4921 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     4981 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     5041 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     5101 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     5161 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     5221 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     5281 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     5341 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     5401 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     5461 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     5521 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     5581 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     5641 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     5701 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     5761 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     5821 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     5881 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     5941 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     6001 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     6061 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     6121 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     6181 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     6241 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     6301 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     6361 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     6421 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     6481 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     6541 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     6601 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     6661 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     6721 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     6781 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     6841 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     6901 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     6961 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     7021 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     7081 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     7141 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     7201 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     7261 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     7321 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     7381 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     7441 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     7501 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     7561 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     7621 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     7681 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     7741 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     7801 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     7861 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     7921 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     7981 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     8041 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     8101 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     8161 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     8221 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     8281 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     8341 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     8401 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     8461 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     8521 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     8581 tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa
     8641 gaaagattac aaagacgatg acgataaggg cagtggagag ggcagaggaa gtctgctaac
     8701 atgcggtgac gtcgaggaga atcctggccc agtgagcaag ggcgaggagc tgttcaccgg
     8761 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     8821 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     8881 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     8941 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     9001 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     9061 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     9121 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     9181 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     9241 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     9301 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     9361 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     9421 tctcggcatg gacgagctgt acaagggatc cggcgcaaca aacttctctc tgctgaaaca
     9481 agccggagat gtcgaagaga atcctggacc gaccgagtac aagcccacgg tgcgcctcgc
     9541 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     9601 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     9661 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     9721 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     9781 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     9841 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc
     9901 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     9961 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
    10021 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
    10081 gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac ctctggatta
    10141 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
    10201 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
    10261 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
    10321 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
    10381 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
    10441 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
    10501 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
    10561 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
    10621 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
    10681 gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag
    10741 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
    10801 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
    10861 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
    10921 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
    10981 ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc
    11041 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
    11101 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
    11161 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
    11221 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
    11281 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
    11341 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
    11401 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
    11461 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
    11521 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
    11581 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
    11641 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
    11701 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
    11761 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
    11821 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
    11881 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
    11941 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
    12001 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
    12061 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
    12121 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
    12181 acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg
    12241 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
    12301 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
    12361 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
    12421 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
    12481 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
    12541 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt
    12601 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
    12661 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
    12721 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
    12781 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
    12841 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
    12901 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
    12961 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
    13021 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
    13081 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
    13141 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
    13201 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
    13261 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
    13321 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
    13381 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
    13441 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
    13501 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
    13561 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
    13621 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
    13681 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
    13741 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
    13801 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
    13861 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
    13921 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
    13981 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
    14041 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
    14101 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
    14161 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
    14221 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
    14281 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
    14341 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
    14401 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
    14461 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
    14521 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
    14581 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
    14641 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
    14701 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
    14761 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
    14821 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
    14881 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
    14941 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
    15001 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
    15061 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
    15121 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
    15181 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
    15241 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
    15301 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
    15361 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
    15421 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
    15481 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
    15541 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
    15601 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
//
