LOCUS       sequence-273891-       13261 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized inactive SpCas9 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 13261)
  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 13261)
  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_09 -
            hU6-BsmBI-sgRNA(E+F)-barcode-EFS-KRAB...
FEATURES             Location/Qualifiers
     source          1..13261
                     /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..2855
                     /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     2778..2797
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3026..3237
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3256..3265
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3292..3477
                     /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             join(3499..3525,3526..3528,3529..6015,6016..6018,
                     6019..7602)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3499..7602
                     /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
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7603..7650
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7651..7674
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7684..7740
                     /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             7741..8337
                     /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     8234..8254
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8353..8941
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8406..8426)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8824..8835)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9013..9246
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9272..9289)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9278..9502
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9548..9976
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9635..9654)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9845..9866
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9985..10005)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9990..10319
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10170..10305
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10232..10251
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10367..10414
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10433..10807
                     /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    10937..11058
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10974..10993)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11028..11047
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11107..11123)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11107..11123)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11120..11142)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11131..11147
                     /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(11155..11161,11162..11179,11180..11185))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11200..11221
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11338..11355)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11509..12097)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11589..11608)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12268..13059,13060..13128))
                     /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     12891..12910
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13129..13233)
                     /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 tcatatttgc
     2641 atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga
     2701 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2761 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2821 tggctttata tatcttgtgg aaaggacgaa acaccggaga cggacgtctc tgtttaagag
     2881 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
     2941 tggcaccgag tcggtgcttt ttttgaattc ggcggcgcta gctaggtctt gaaaggagtg
     3001 ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     3061 ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg
     3121 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     3181 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggac
     3241 cggttctaga gcgctgccac catggatgct aagtcactaa ctgcctggtc ccggacactg
     3301 gtgaccttca aggatgtatt tgtggacttc accagggagg agtggaagct gctggacact
     3361 gctcagcaga tcgtgtacag aaatgtgatg ctggagaact ataagaacct ggtttccttg
     3421 ggttatcagc ttactaagcc agatgtgatc ctccggttgg agaagggaga agagcccggc
     3481 ggttccggcg gagggtcgat ggacaagaag tacagcatcg gcctggccat cggcaccaac
     3541 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
     3601 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggcgccct gctgttcgac
     3661 agcggagaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga
     3721 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
     3781 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
     3841 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
     3901 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
     3961 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
     4021 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
     4081 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
     4141 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
     4201 aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
     4261 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
     4321 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
     4381 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     4441 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     4501 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     4561 agcaagaacg gctacgccgg ctacatcgat ggcggagcca gccaggaaga gttctacaag
     4621 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     4681 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     4741 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     4801 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     4861 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     4921 ccctggaact tcgaggaagt ggtggacaag ggcgccagcg cccagagctt catcgagcgg
     4981 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     5041 tacgagtact tcaccgtgta caacgagctg accaaagtga aatacgtgac cgagggaatg
     5101 agaaagcccg ccttcctgag cggcgagcag aaaaaagcca tcgtggacct gctgttcaag
     5161 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     5221 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     5281 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     5341 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     5401 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     5461 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     5521 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     5581 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     5641 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     5701 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     5761 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     5821 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     5881 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     5941 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     6001 gactacgatg tggacgctat cgtgcctcag agctttctga aggacgactc catcgataac
     6061 aaagtgctga ctcggagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag
     6121 gtcgtgaaga agatgaagaa ctactggcgc cagctgctga atgccaagct gattacccag
     6181 aggaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     6241 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     6301 ctggactccc ggatgaacac taagtacgac gagaacgaca aactgatccg ggaagtgaaa
     6361 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     6421 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     6481 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag
     6541 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     6601 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     6661 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaacaggcga gatcgtgtgg
     6721 gataagggcc gggactttgc caccgtgcgg aaagtgctgt ctatgcccca agtgaatatc
     6781 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     6841 aacagcgaca agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     6901 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     6961 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     7021 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     7081 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     7141 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     7201 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     7261 aaacagctgt ttgtggaaca gcacaaacac tacctggacg agatcatcga gcagatcagc
     7321 gagttctcca agagagtgat cctggccgac gctaatctgg acaaggtgct gagcgcctac
     7381 aacaagcaca gagacaagcc tatcagagag caggccgaga atatcatcca cctgtttacc
     7441 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     7501 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     7561 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaagcgacc tgccgccaca
     7621 aagaaggctg gacaggctaa gaagaagaaa gattacaaag acgatgacga taagggatcc
     7681 ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg
     7741 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     7801 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     7861 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     7921 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     7981 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     8041 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     8101 gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg tctgggcagc
     8161 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     8221 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     8281 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg
     8341 cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     8401 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     8461 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     8521 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     8581 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     8641 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     8701 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     8761 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     8821 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     8881 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     8941 cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     9001 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     9061 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     9121 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     9181 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     9241 ctagcagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     9301 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     9361 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     9421 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     9481 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     9541 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     9601 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     9661 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     9721 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     9781 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     9841 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     9901 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     9961 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
    10021 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
    10081 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
    10141 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
    10201 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
    10261 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
    10321 agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc
    10381 atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa
    10441 gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg
    10501 gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg
    10561 ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct
    10621 ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc
    10681 cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg
    10741 gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca
    10801 ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
    10861 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
    10921 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
    10981 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
    11041 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
    11101 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
    11161 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
    11221 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
    11281 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
    11341 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
    11401 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
    11461 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
    11521 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
    11581 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
    11641 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
    11701 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
    11761 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
    11821 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
    11881 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
    11941 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
    12001 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
    12061 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
    12121 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
    12181 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
    12241 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
    12301 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
    12361 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
    12421 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
    12481 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
    12541 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
    12601 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
    12661 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
    12721 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
    12781 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
    12841 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
    12901 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
    12961 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
    13021 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
    13081 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
    13141 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
    13201 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
    13261 c
//
