LOCUS       sequence-376995-       13142 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG tagged SpdCas9 KRAB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13142)
  AUTHORS   Escobar M, Li J, Patel A, Liu S, Xu Q, Hilton IB
  TITLE     Quantification of Genome Editing and Transcriptional Control 
            Capabilities Reveals Hierarchies among Diverse CRISPR/Cas Systems in
            Human Cells.
  JOURNAL   ACS Synth Biol. 2022 Oct 21;11(10):3239-3250. doi: 
            10.1021/acssynbio.2c00156. Epub 2022 Sep 26.
  PUBMED    36162812
REFERENCE   2  (bases 1 to 13142)
  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: 
            "10.1021/acssynbio.2c00156"; journalName: "ACS Synth Biol"; date: 
            "2022-10-21- 21"; volume: "11"; issue: "10"; pages: "3239-3250"
COMMENT     Sequence Label: SpdCas9 KRAB
FEATURES             Location/Qualifiers
     source          1..13142
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1062..1295
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1480..1524
                     /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    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        2071..2146
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2208..2419
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2438..2447
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2444..2470,2471..2473,2474..4960,4961..4963,
                     4964..6547)
                     /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             2444..6547
                     /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             6548..6595
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6596..6619
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6653..6847
                     /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="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             6914..6934
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6947..7003
                     /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             7004..7600
                     /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     7497..7517
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7616..8204
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7669..7689)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8087..8098)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8276..8509
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8535..8552)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8541..8765
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8811..9239
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8898..8917)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9108..9129
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9248..9268)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9253..9582
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9433..9568
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9495..9514
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9630..9677
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9696..10070
                     /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    10200..10321
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10237..10256)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10291..10310
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10370..10386)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10370..10386)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10383..10405)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10394..10410
                     /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(10418..10424,10425..10442,10443..10448))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10463..10484
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10601..10618)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10772..11360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10852..10871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11531..12322,12323..12391))
                     /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     12154..12173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12392..12496)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12571..12590)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12762..13141
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
      961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
     1981 taaggtaccg atatcactag tgttccatta actgcgctaa aaataatttt taaatctttt
     2041 ttagcttctt gctctttttt gtacgtctct gttttagagc tagaaatagc aagttaaaat
     2101 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcgc
     2161 tagctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac
     2221 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg gtgcctagag
     2281 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     2341 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     2401 gtttgccgcc agaacacagg accggttcta gagcgctgcc accatggaca agaagtacag
     2461 catcggcctg gccatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa
     2521 ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa
     2581 cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag
     2641 aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt
     2701 cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag agtccttcct
     2761 ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt
     2821 ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac
     2881 cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg
     2941 ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat
     3001 ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt
     3061 ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat
     3121 cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct
     3181 gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct
     3241 gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta
     3301 cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct
     3361 gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga
     3421 cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa
     3481 gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg
     3541 agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac
     3601 cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga
     3661 caacggcagc atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca
     3721 ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt
     3781 ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac
     3841 cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc
     3901 ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa
     3961 ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa
     4021 agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa
     4081 ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga
     4141 ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg
     4201 gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt
     4261 cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt
     4321 tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa
     4381 agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct
     4441 gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga
     4501 cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga
     4561 ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa
     4621 tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga
     4681 gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga
     4741 gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga
     4801 gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct
     4861 gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca
     4921 ggaactggac atcaaccggc tgtccgacta cgatgtggac gccatcgtgc ctcagagctt
     4981 tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa
     5041 gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct
     5101 gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg
     5161 cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca
     5221 gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa
     5281 tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt
     5341 ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga
     5401 cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag
     5461 cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga
     5521 gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt
     5581 caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa
     5641 cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt
     5701 gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag
     5761 caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg
     5821 ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt
     5881 ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat
     5941 caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg
     6001 ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct
     6061 ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact
     6121 ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa
     6181 gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct
     6241 ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa
     6301 tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc
     6361 cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta
     6421 ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac
     6481 cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg
     6541 aggcgacaag cgacctgccg ccacaaagaa ggctggacag gctaagaaga agaaagatta
     6601 caaagacgat gacgataagg gatccgatgc taagtcactg actgcctggt cccggacact
     6661 ggtgaccttc aaggatgtgt ttgtggactt caccagggag gagtggaagc tgctggacac
     6721 tgctcagcag atcctgtaca gaaatgtgat gctggagaac tataagaacc tggtttcctt
     6781 gggttatcag cttactaagc cagatgtgat cctccggttg gagaagggag aagagccctg
     6841 gctggtggag agagaaattc accaagagac ccatcctgat tcagagactg catttgaaat
     6901 caaatcatca gttccgaaaa agaaacgcaa agttgctagc tccggcgcaa caaacttctc
     6961 tctgctgaaa caagccggag atgtcgaaga gaatcctgga ccgaccgagt acaagcccac
     7021 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     7081 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     7141 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     7201 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     7261 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     7321 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     7381 cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     7441 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     7501 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     7561 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca
     7621 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     7681 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     7741 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     7801 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     7861 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     7921 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     7981 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     8041 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     8101 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     8161 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca
     8221 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     8281 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
     8341 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     8401 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     8461 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag ggcccgttta
     8521 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     8581 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     8641 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     8701 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     8761 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     8821 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     8881 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     8941 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     9001 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     9061 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     9121 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     9181 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     9241 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     9301 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     9361 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     9421 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     9481 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     9541 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     9601 atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg
     9661 gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc
     9721 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt
     9781 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt
     9841 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc
     9901 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg
     9961 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc
    10021 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac
    10081 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
    10141 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
    10201 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
    10261 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
    10321 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
    10381 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
    10441 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
    10501 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
    10561 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
    10621 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
    10681 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
    10741 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
    10801 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
    10861 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
    10921 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
    10981 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
    11041 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
    11101 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
    11161 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
    11221 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
    11281 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
    11341 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
    11401 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
    11461 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
    11521 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
    11581 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
    11641 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
    11701 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
    11761 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
    11821 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
    11881 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
    11941 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
    12001 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
    12061 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
    12121 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
    12181 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
    12241 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
    12301 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
    12361 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
    12421 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
    12481 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
    12541 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
    12601 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
    12661 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
    12721 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
    12781 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
    12841 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
    12901 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
    12961 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
    13021 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
    13081 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
    13141 gg
//
