LOCUS       sequence-254680-       13011 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of Cas9 and a control gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13011)
  AUTHORS   Gao W, Li W, Xiao T, Liu XS, Kaelin WG Jr
  TITLE     Inactivation of the PBRM1 tumor suppressor gene amplifies the 
            HIF-response in VHL-/- clear cell renal carcinoma.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):1027-1032. doi: 
            10.1073/pnas.1619726114. Epub 2017 Jan 12.
  PUBMED    28082722
REFERENCE   2  (bases 1 to 13011)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.1619726114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-01-31- 31"; volume: "114"; issue: "5"; pages: 
            "1027-1032"
COMMENT     Sequence Label: sgCtr- LentiCRISPRv2
FEATURES             Location/Qualifiers
     source          1..13011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    261..378
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        429..677
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     429..449
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     600..619
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        697..772
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        834..1045
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1064..1073
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1070..5173
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1070..5173
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5174..5221
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5222..5245
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5255..5311
                     /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             5312..5908
                     /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     5805..5825
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5924..6512
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5977..5997)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6395..6406)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6584..6817
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6843..6860)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6849..7073
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7119..7547
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7206..7225)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7416..7437
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7556..7576)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7561..7890
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7741..7876
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7803..7822
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7938..7985
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8004..8378
                     /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    8508..8629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8545..8564)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8599..8618
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8678..8694)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8678..8694)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8691..8713)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8702..8718
                     /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(8726..8732,8733..8750,8751..8756))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8771..8792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8909..8926)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9080..9668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9160..9179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9839..10630,10631..10699))
                     /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     10462..10481
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10700..10804)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10879..10898)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        11070..11449
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11451..11649
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11604..11624
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11667..11847
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11894..12019
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12512..12745
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12930..12974
                     /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"
ORIGIN
        1 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
       61 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
      121 caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca
      181 gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa
      241 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
      301 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
      361 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt ttggttaatt
      421 aaggtaccga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
      481 ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt
      541 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
      601 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
      661 ggaaaggacg aaacaccgct tccgcggccc gttcaagttt tagagctaga aatagcaagt
      721 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttga
      781 attcgctagc taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga
      841 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc
      901 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
      961 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     1021 caacgggttt gccgccagaa cacaggaccg gttctagagc gctgccacca tggacaagaa
     1081 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     1141 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     1201 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     1261 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     1321 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     1381 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     1441 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     1501 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     1561 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     1621 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     1681 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     1741 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     1801 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     1861 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     1921 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     1981 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     2041 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     2101 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     2161 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     2221 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     2281 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     2341 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     2401 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     2461 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     2521 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     2581 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     2641 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     2701 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     2761 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     2821 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     2881 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     2941 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     3001 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     3061 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     3121 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     3181 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     3241 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     3301 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     3361 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     3421 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     3481 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     3541 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     3601 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     3661 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     3721 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     3781 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     3841 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     3901 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     3961 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     4021 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     4081 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     4141 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     4201 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     4261 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     4321 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     4381 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     4441 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     4501 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     4561 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     4621 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     4681 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     4741 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     4801 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     4861 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     4921 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     4981 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     5041 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     5101 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     5161 gctgggaggc gacaagcgac ctgccgccac aaagaaggct ggacaggcta agaagaagaa
     5221 agattacaaa gacgatgacg ataagggatc cggcgcaaca aacttctctc tgctgaaaca
     5281 agccggagat gtcgaagaga atcctggacc gaccgagtac aagcccacgg tgcgcctcgc
     5341 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     5401 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     5461 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     5521 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     5581 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     5641 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc
     5701 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     5761 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     5821 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     5881 gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac ctctggatta
     5941 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     6001 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     6061 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     6121 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     6181 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     6241 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     6301 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     6361 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     6421 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     6481 gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag
     6541 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     6601 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     6661 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     6721 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     6781 ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc
     6841 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     6901 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     6961 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     7021 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     7081 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     7141 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     7201 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     7261 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     7321 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     7381 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     7441 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     7501 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     7561 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     7621 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     7681 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     7741 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     7801 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     7861 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     7921 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
     7981 acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg
     8041 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     8101 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     8161 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     8221 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     8281 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     8341 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt
     8401 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     8461 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     8521 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     8581 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     8641 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     8701 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     8761 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     8821 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     8881 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     8941 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     9001 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     9061 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     9121 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     9181 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     9241 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     9301 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     9361 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     9421 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     9481 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     9541 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     9601 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     9661 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     9721 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     9781 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     9841 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     9901 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     9961 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
    10021 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
    10081 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
    10141 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
    10201 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
    10261 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
    10321 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
    10381 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
    10441 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
    10501 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
    10561 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
    10621 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
    10681 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
    10741 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
    10801 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
    10861 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
    10921 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
    10981 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
    11041 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
    11101 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
    11161 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
    11221 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
    11281 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
    11341 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
    11401 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
    11461 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
    11521 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
    11581 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
    11641 tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg
    11701 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
    11761 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
    11821 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
    11881 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
    11941 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
    12001 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
    12061 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
    12121 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
    12181 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
    12241 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
    12301 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
    12361 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
    12421 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
    12481 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
    12541 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
    12601 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
    12661 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
    12721 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
    12781 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
    12841 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
    12901 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
    12961 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat a
//
