LOCUS       sequence-394872-       13124 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpCas9 construct to knockout murine Cd274.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13124)
  AUTHORS   Ruf B, Bruhns M, Babaei S, Kedei N, Ma L, Revsine M, Benmebarek MR, 
            Ma C, Heinrich B, Subramanyam V, Qi J, Wabitsch S, Green BL, Bauer 
            KC, Myojin Y, Greten LT, McCallen JD, Huang P, Trehan R, Wang X, Nur
            A, Murphy Soika DQ, Pouzolles M, Evans CN, Chari R, Kleiner DE, 
            Telford W, Dadkhah K, Ruchinskas A, Stovroff MK, Kang J, Oza K, 
            Ruchirawat M, Kroemer A, Wang XW, Claassen M, Korangy F, Greten TF
  TITLE     Tumor-associated macrophages trigger MAIT cell dysfunction at the 
            HCC invasive margin.
  JOURNAL   Cell. 2023 Aug 17;186(17):3686-3705.e32. doi: 
            10.1016/j.cell.2023.07.026.
  PUBMED    37595566
REFERENCE   2  (bases 1 to 13124)
  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.cell.2023.07"; journalName: "Cell"; date: "2023-08-17- 
            17"; volume: "186"; issue: "17"; pages: "3686-3705.e32"
COMMENT     Sequence Label: pGMC00027
FEATURES             Location/Qualifiers
     source          1..13124
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(55..74)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        246..625
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        627..825
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     780..800
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             843..1023
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1070..1195
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1688..1921
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2106..2150
                     /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    2448..2565
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2616..2864
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2616..2636
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2787..2806
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2886..2961
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3023..3234
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3253..3262
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3259..7362
                     /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             3259..7362
                     /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             7363..7410
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7411..7434
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7444..7500
                     /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             7501..8208
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(7643..7661)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7911..7930
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    8224..8812
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8277..8297)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8695..8706)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8884..9117
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9143..9160)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9149..9373
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9419..9847
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9506..9525)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9716..9737
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9856..9876)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9861..10190
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10041..10176
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10103..10122
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10238..10285
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10304..10678
                     /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    10808..10929
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10845..10864)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10899..10918
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10978..10994)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10978..10994)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10991..11013)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11002..11018
                     /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(11026..11032,11033..11050,11051..11056))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11071..11092
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11209..11226)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11380..11968)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11460..11479)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12139..12930,12931..12999))
                     /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     12762..12781
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13000..13104)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
       61 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
      121 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
      181 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
      241 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
      301 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      361 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      421 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      481 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      541 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      601 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      661 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      721 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      781 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt tttgcctgta
      841 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
      901 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
      961 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     1021 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg
     1081 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac
     1141 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat
     1201 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa
     1261 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
     1321 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc
     1381 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
     1441 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga
     1501 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag
     1561 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac
     1621 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag
     1681 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag
     1741 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga
     1801 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca
     1861 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc
     1921 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta
     1981 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca
     2041 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc
     2101 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt
     2161 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg
     2221 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt
     2281 caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa
     2341 tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg aacggatcgg
     2401 cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt taaaagaaaa
     2461 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     2521 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     2581 gacagcagag atccagtttg gttaattaag gtaccgaggg cctatttccc atgattcctt
     2641 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     2701 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     2761 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     2821 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggctc caaaggactt
     2881 gtacggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     2941 aaagtggcac cgagtcggtg cttttttgaa ttcgctagct aggtcttgaa aggagtggga
     3001 attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
     3061 gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa
     3121 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
     3181 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggaccgg
     3241 ttctagagcg ctgccaccat ggacaagaag tacagcatcg gcctggacat cggcaccaac
     3301 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
     3361 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac
     3421 agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga
     3481 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
     3541 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
     3601 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
     3661 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
     3721 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
     3781 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
     3841 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
     3901 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
     3961 aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
     4021 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
     4081 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
     4141 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     4201 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     4261 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     4321 agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag
     4381 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     4441 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     4501 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     4561 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     4621 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     4681 ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg
     4741 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     4801 tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg
     4861 agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag
     4921 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     4981 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     5041 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     5101 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     5161 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     5221 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     5281 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     5341 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     5401 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     5461 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     5521 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     5581 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     5641 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     5701 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     5761 gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac
     5821 aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag
     5881 gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag
     5941 agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     6001 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     6061 ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa
     6121 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     6181 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     6241 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag
     6301 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     6361 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     6421 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     6481 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     6541 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     6601 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     6661 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     6721 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     6781 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     6841 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     6901 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     6961 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     7021 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     7081 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     7141 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     7201 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     7261 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     7321 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaagcgacc tgccgccaca
     7381 aagaaggctg gacaggctaa gaagaagaaa gattacaaag acgatgacga taagggatcc
     7441 ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg
     7501 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     7561 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     7621 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     7681 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     7741 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     7801 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     7861 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     7921 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     7981 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     8041 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     8101 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     8161 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaaac gcgttaagtc
     8221 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     8281 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     8341 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     8401 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     8461 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     8521 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     8581 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     8641 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     8701 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     8761 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     8821 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     8881 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     8941 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     9001 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     9061 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg
     9121 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     9181 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     9241 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     9301 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     9361 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     9421 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     9481 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     9541 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     9601 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     9661 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     9721 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     9781 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     9841 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     9901 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     9961 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
    10021 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
    10081 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
    10141 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
    10201 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata
    10261 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag
    10321 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
    10381 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
    10441 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
    10501 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
    10561 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
    10621 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
    10681 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
    10741 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
    10801 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
    10861 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
    10921 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
    10981 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
    11041 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
    11101 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
    11161 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
    11221 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
    11281 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
    11341 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
    11401 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
    11461 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
    11521 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
    11581 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
    11641 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
    11701 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
    11761 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
    11821 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
    11881 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
    11941 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
    12001 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
    12061 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
    12121 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
    12181 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
    12241 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
    12301 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
    12361 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
    12421 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
    12481 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
    12541 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
    12601 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
    12661 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
    12721 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
    12781 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
    12841 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
    12901 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
    12961 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
    13021 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
    13081 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgc
//
