LOCUS       sequence-273882-       13025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized SpCas9 nuclease in mammalian cells. 
            For cloning of sgRNAs using BsmBI. Contains a unique barcode 
            downstream of sgRNA cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13025)
  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 13025)
  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_01 -
            hU6-BsmBI-sgRNA(E+F)-barcode-EFS-Cas9...
FEATURES             Location/Qualifiers
     source          1..13025
                     /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             3263..7366
                     /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             3263..7366
                     /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             7367..7414
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7415..7438
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7448..7504
                     /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             7505..8101
                     /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     7998..8018
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8117..8705
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8170..8190)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8588..8599)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8777..9010
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9036..9053)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9042..9266
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9312..9740
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9399..9418)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9609..9630
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9749..9769)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9754..10083
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9934..10069
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9996..10015
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10131..10178
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10197..10571
                     /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    10701..10822
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10738..10757)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10792..10811
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10871..10887)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10871..10887)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10884..10906)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10895..10911
                     /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(10919..10925,10926..10943,10944..10949))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10964..10985
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11102..11119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11273..11861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11353..11372)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12032..12823,12824..12892))
                     /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     12655..12674
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12893..12997)
                     /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 ctaatgggct 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 ccatggacaa gaagtacagc atcggcctgg acatcggcac
     3301 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     3361 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
     3421 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     3481 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
     3541 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     3601 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     3661 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     3721 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     3781 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     3841 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     3901 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     3961 gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     4021 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     4081 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     4141 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     4201 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     4261 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     4321 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     4381 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     4441 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     4501 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     4561 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     4621 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     4681 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     4741 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     4801 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     4861 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt
     4921 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
     4981 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
     5041 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
     5101 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
     5161 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
     5221 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
     5281 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
     5341 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
     5401 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
     5461 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
     5521 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
     5581 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
     5641 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
     5701 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
     5761 gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga
     5821 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga
     5881 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
     5941 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
     6001 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
     6061 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
     6121 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
     6181 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
     6241 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
     6301 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
     6361 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
     6421 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
     6481 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
     6541 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
     6601 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
     6661 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
     6721 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
     6781 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
     6841 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
     6901 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
     6961 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
     7021 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
     7081 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
     7141 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
     7201 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
     7261 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
     7321 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaagc gacctgccgc
     7381 cacaaagaag gctggacagg ctaagaagaa gaaagattac aaagacgatg acgataaggg
     7441 atccggcgca acaaacttct ctctgctgaa acaagccgga gatgtcgaag agaatcctgg
     7501 accgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     7561 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     7621 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     7681 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     7741 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     7801 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     7861 gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg
     7921 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     7981 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     8041 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     8101 aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     8161 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     8221 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     8281 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     8341 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     8401 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     8461 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     8521 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     8581 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     8641 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     8701 cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     8761 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt
     8821 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     8881 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     8941 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     9001 atctctagca gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     9061 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     9121 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     9181 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     9241 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     9301 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     9361 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     9421 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     9481 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     9541 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     9601 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     9661 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     9721 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     9781 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     9841 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     9901 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     9961 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
    10021 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
    10081 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt
    10141 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg
    10201 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt
    10261 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg
    10321 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca
    10381 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg
    10441 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt
    10501 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg
    10561 agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg
    10621 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
    10681 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
    10741 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
    10801 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
    10861 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
    10921 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
    10981 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
    11041 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
    11101 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
    11161 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
    11221 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
    11281 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
    11341 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
    11401 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
    11461 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
    11521 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
    11581 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
    11641 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
    11701 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
    11761 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
    11821 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
    11881 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
    11941 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
    12001 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
    12061 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
    12121 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
    12181 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
    12241 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
    12301 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
    12361 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
    12421 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
    12481 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
    12541 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
    12601 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
    12661 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
    12721 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
    12781 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
    12841 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
    12901 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
    12961 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
    13021 ctgac
//
