LOCUS       sequence-269072-       13262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized inactive xCas9 3.7 fused to a 
            transcriptional repressor KRAB in mammalian cells. For cloning of 
            sgRNAs using BsmBI. Contains a barcode downstream of sgRNA 
            cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13262)
  AUTHORS   Legut M, Daniloski Z, Xue X, McKenzie D, Guo X, Wessels HH, Sanjana 
            NE
  TITLE     High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene 
            Knockout and Transcriptional Modulation.
  JOURNAL   Cell Rep. 2020 Mar 3;30(9):2859-2868.e5. doi: 
            10.1016/j.celrep.2020.02.010.
  PUBMED    32130891
REFERENCE   2  (bases 1 to 13262)
  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: 
            "10.1016/j.celrep.2020.02"; journalName: "Cell Rep"; date: 
            "2020-03-3- 3"; volume: "30"; issue: "9"; pages: "2859-2868.e5"
COMMENT     Sequence Label: pCC_11 -
            hU6-BsmBI-sgRNA(E+F)-barcode-EFS-KRAB...
FEATURES             Location/Qualifiers
     source          1..13262
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2608..2856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2608..2628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2779..2798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3027..3238
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3257..3266
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3293..3478
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             3500..7603
                     /codon_start=1
                     /product="catalytically dead Streptococcus pyogenes Cas9 
                     mutant with broad PAM compatibility (Hu et al., 2018)"
                     /label=dxCas9(3.7)
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3500..7603
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7604..7651
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7652..7675
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7685..7741
                     /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             7742..8338
                     /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     8235..8255
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8354..8942
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8407..8427)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8825..8836)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9014..9247
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9273..9290)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9279..9503
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9549..9977
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9636..9655)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9846..9867
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9986..10006)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9991..10320
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10171..10306
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10233..10252
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10368..10415
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10434..10808
                     /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    10938..11059
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10975..10994)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11029..11048
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11108..11124)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11108..11124)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11121..11143)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11132..11148
                     /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(11156..11162,11163..11180,11181..11186))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11201..11222
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11339..11356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11510..12098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11590..11609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12269..13060,13061..13129))
                     /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     12892..12911
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13130..13234)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacaccggag acggacgtct ctgtttaaga
     2881 gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa
     2941 gtggcaccga gtcggtgctt tttttgaatt catgtaggct agctaggtct tgaaaggagt
     3001 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     3061 gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg
     3121 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     3181 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga
     3241 ccggttctag agcgctgcca ccatggatgc taagtcacta actgcctggt cccggacact
     3301 ggtgaccttc aaggatgtat ttgtggactt caccagggag gagtggaagc tgctggacac
     3361 tgctcagcag atcgtgtaca gaaatgtgat gctggagaac tataagaacc tggtttcctt
     3421 gggttatcag cttactaagc cagatgtgat cctccggttg gagaagggag aagagcccgg
     3481 cggttccggc ggagggtcga tggacaagaa gtacagcatc ggcctggcca tcggcaccaa
     3541 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt
     3601 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga
     3661 cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag
     3721 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga
     3781 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga
     3841 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac
     3901 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat
     3961 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct
     4021 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca
     4081 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc
     4141 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa
     4201 gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag
     4261 caacttcgac ctggccgagg ataccaaact gcagctgagc aaggacacct acgacgacga
     4321 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa
     4381 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa
     4441 ggcccccctg agcgcctcta tgatcaagct gtacgacgag caccaccagg acctgaccct
     4501 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca
     4561 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa
     4621 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa
     4681 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcatcatcc cccaccagat
     4741 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa
     4801 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc
     4861 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac
     4921 cccctggaac ttcgagaagg tggtggacaa gggcgcttcc gcccagagct tcatcgagcg
     4981 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct
     5041 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat
     5101 gagaaagccc gccttcctga gcggcgacca gaaaaaggcc atcgtggacc tgctgttcaa
     5161 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg
     5221 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata
     5281 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga
     5341 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga
     5401 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg
     5461 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca
     5521 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat
     5581 ccagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc
     5641 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa
     5701 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca
     5761 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca
     5821 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca
     5881 gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta
     5941 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc
     6001 cgactacgat gtggacgcta tcgtgcctca gagctttctg aaggacgact ccatcgacaa
     6061 caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga
     6121 ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca
     6181 gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc
     6241 cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat
     6301 cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa
     6361 agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa
     6421 agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg
     6481 aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa
     6541 ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc
     6601 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa
     6661 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg
     6721 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat
     6781 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag
     6841 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt
     6901 cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc
     6961 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt
     7021 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct
     7081 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct
     7141 ggcctctgcc ggcgtgctgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa
     7201 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca
     7261 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag
     7321 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta
     7381 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac
     7441 cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa
     7501 gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg
     7561 cctgtacgag acacggatcg acctgtctca gctgggaggc gacaagcgac ctgccgccac
     7621 aaagaaggct ggacaggcta agaagaagaa agattacaaa gacgatgacg ataagggatc
     7681 cggcgcaaca aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc
     7741 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     7801 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     7861 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     7921 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     7981 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     8041 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     8101 cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag
     8161 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     8221 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     8281 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac
     8341 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     8401 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     8461 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     8521 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     8581 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     8641 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     8701 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     8761 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     8821 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     8881 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     8941 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     9001 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
     9061 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     9121 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     9181 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     9241 tctagcaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     9301 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     9361 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     9421 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     9481 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     9541 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     9601 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     9661 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     9721 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     9781 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     9841 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     9901 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     9961 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
    10021 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
    10081 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
    10141 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
    10201 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
    10261 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
    10321 aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat
    10381 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca
    10441 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct
    10501 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc
    10561 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc
    10621 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt
    10681 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg
    10741 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc
    10801 aggactgaca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
    10861 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
    10921 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
    10981 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
    11041 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
    11101 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
    11161 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
    11221 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
    11281 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
    11341 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
    11401 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
    11461 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
    11521 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
    11581 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
    11641 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
    11701 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
    11761 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
    11821 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
    11881 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
    11941 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
    12001 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
    12061 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
    12121 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
    12181 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
    12241 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
    12301 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
    12361 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
    12421 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
    12481 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
    12541 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
    12601 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
    12661 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
    12721 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
    12781 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
    12841 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
    12901 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
    12961 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
    13021 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
    13081 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
    13141 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
    13201 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
    13261 ac
//
