LOCUS       sequence-212211-       13128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of Cas9 and gRNA targeting PBRM1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13128)
  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 13128)
  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: sgPBRM1-2- LentiCRISPRv2
FEATURES             Location/Qualifiers
     source          1..13128
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    187..304
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    370..394
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     557..576
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        654..729
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(775..799)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        877..1088
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1107..1116
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1113..5216
                     /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             1113..5216
                     /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             5217..5264
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5265..5288
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5298..5354
                     /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             5355..5951
                     /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     5848..5868
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5967..6555
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6020..6040)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6438..6449)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6627..6860
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6886..6903)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6892..7116
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7162..7590
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7249..7268)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7459..7480
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7599..7619)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7604..7933
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7784..7919
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7846..7865
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7981..8028
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8047..8421
                     /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    8551..8672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8588..8607)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8642..8661
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8721..8737)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8721..8737)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8734..8756)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8745..8761
                     /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(8769..8775,8776..8793,8794..8799))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8814..8835
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8952..8969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9123..9711)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9203..9222)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9882..10673,10674..10742))
                     /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     10505..10524
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10743..10847)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10922..10941)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        11113..11492
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11494..11692
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11647..11667
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11710..11890
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11937..12062
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12555..12788
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12973..13017
                     /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 agagttaggc agggatattc accattatcg tttcagaccc acctcccaac cccgagggga
       61 cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt
      121 cgattagtga acggatcggc actgcgtgcg ccaattctgc agacaaatgg cagtattcat
      181 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
      241 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
      301 ttttcgggtt tattacaggg acagcagaga tccagtttgg ttaattaacc cacaaggaga
      361 cgaccttcca caagtttgta caaaaaagca ggcttttccc atgattcctt catatttgca
      421 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
      481 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
      541 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
      601 ggctttatat atcttgtgga aaggacgaaa caccgcatcc ttatagtctc ggagttttag
      661 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      721 agtcggtgct tttttgaatt ctagatcttg agacaaatgg cagtattcat ccacacccag
      781 ctttcttgta caaagtggtg ggtgaggcgg ttcaggtggc ggaggaagct agctaggtct
      841 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
      901 tccccgagaa gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg
      961 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     1021 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     1081 gaacacagga ccggttctag agcgctgcca ccatggacaa gaagtacagc atcggcctgg
     1141 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
     1201 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
     1261 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
     1321 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
     1381 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
     1441 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
     1501 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     1561 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     1621 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
     1681 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     1741 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     1801 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc
     1861 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     1921 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     1981 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     2041 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     2101 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     2161 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
     2221 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     2281 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     2341 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     2401 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     2461 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     2521 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
     2581 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     2641 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
     2701 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
     2761 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     2821 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct
     2881 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg
     2941 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag
     3001 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc
     3061 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca
     3121 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca
     3181 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga
     3241 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca
     3301 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag
     3361 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca
     3421 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag
     3481 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga
     3541 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca
     3601 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg
     3661 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg
     3721 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca
     3781 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg
     3841 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc
     3901 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga
     3961 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt
     4021 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga
     4081 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt
     4141 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg
     4201 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga
     4261 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg
     4321 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc
     4381 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta
     4441 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga
     4501 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg
     4561 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg
     4621 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag
     4681 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc
     4741 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct
     4801 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg
     4861 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca
     4921 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag
     4981 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca
     5041 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca
     5101 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc
     5161 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaagc
     5221 gacctgccgc cacaaagaag gctggacagg ctaagaagaa gaaagattac aaagacgatg
     5281 acgataaggg atccggcgca acaaacttct ctctgctgaa acaagccgga gatgtcgaag
     5341 agaatcctgg accgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     5401 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     5461 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     5521 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     5581 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     5641 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     5701 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca
     5761 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     5821 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     5881 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     5941 ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     6001 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     6061 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     6121 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     6181 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     6241 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     6301 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     6361 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     6421 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     6481 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     6541 gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag
     6601 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     6661 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     6721 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     6781 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     6841 agtgtggaaa atctctagca gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     6901 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     6961 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     7021 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     7081 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     7141 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     7201 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     7261 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     7321 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     7381 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     7441 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     7501 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     7561 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     7621 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     7681 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     7741 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     7801 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     7861 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     7921 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
     7981 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     8041 taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga
     8101 gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc
     8161 gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg
     8221 ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg
     8281 tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc
     8341 gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc
     8401 gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat
     8461 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     8521 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     8581 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     8641 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     8701 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     8761 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     8821 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     8881 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     8941 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     9001 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     9061 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     9121 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     9181 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     9241 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     9301 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     9361 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     9421 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     9481 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     9541 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     9601 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     9661 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     9721 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     9781 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     9841 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     9901 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     9961 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
    10021 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
    10081 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
    10141 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
    10201 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
    10261 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
    10321 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
    10381 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
    10441 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
    10501 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
    10561 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
    10621 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
    10681 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
    10741 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
    10801 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
    10861 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
    10921 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
    10981 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
    11041 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
    11101 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
    11161 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
    11221 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
    11281 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
    11341 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
    11401 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
    11461 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
    11521 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
    11581 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
    11641 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt
    11701 gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
    11761 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
    11821 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
    11881 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
    11941 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
    12001 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
    12061 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
    12121 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
    12181 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
    12241 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
    12301 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
    12361 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct
    12421 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
    12481 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
    12541 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
    12601 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
    12661 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
    12721 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
    12781 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
    12841 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
    12901 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
    12961 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
    13021 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
    13081 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaat
//
