LOCUS       SLEEK-GAPDH-sgRN        9232 bp DNA     circular SYN 28-APR-2025
DEFINITION  Cas9 from S. pyogenes with 2A-EGFP, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9232)
  AUTHORS   Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F
  TITLE     Genome engineering using the CRISPR-Cas9 system.
  JOURNAL   Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. 
            Epub 2013 Oct 24.
  PUBMED    24157548
REFERENCE   2  (bases 1 to 9232)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 14, 2025 from SnapGene 7.1.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nprot.2013.143"; journalName: "Nat Protoc"; date: 
            "2013-11"; volume: "8"; issue: "11"; pages: "2281-308"
COMMENT     Sequence Label: pSpCas9(BB)-2A-GFP (PX458)
FEATURES             Location/Qualifiers
     source          1..9232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          251..270
                     /chromosome="12"
                     /mol_type="genomic DNA"
                     /note="color: #ffffff"
                     /db_xref="taxon:9606"
                     /organism="Homo sapiens"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     129..151
                     /label=Ubi-Lib F
                     /note="color: black; sequence: atttcttgggtagtttgcagttt; 
                     added: 2025-04-28"
     misc_feature    246..250
                     /label=SLEEK-GAPDH-sgRNA
                     /note="TTTGGGATTCCCTTCAGAGA 
                     Efficiency: 51.31, 45% GCself-complementarity: 1
                     MM0: 1
                     MM1: 0
                     MM2: 1
                     MM3: 12"
                     /note="color: #99cc00"
     gene            251..270
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /label=GAPDH
                     /note="glyceraldehyde-3-phosphate dehydrogenase; Derived
                     by automated computational analysis using gene prediction 
                     method: BestRefSeq."
                     /note="color: #a6acb3; direction: RIGHT"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
     misc_feature    251..270
                     /label=sgRNA
                     /note="color: #ff0000"
     primer_bind     251..270
                     /label=SLEEK-GAPDH-sgRNA
                     /note="color: black; sequence: tctaggtatgacaacgaatt"
     primer_bind     complement(251..270)
                     /label=sgRNA'
                     /note="color: black; sequence: aattcgttgtcatacctaga"
     misc_feature    251..267
                     /label=5'arm
                     /note="color: #ff6600"
     mRNA            256..270
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase,
                     transcript variant 4"
                     /label=GAPDH mRNA
                     /note="Derived by automated computational analysis using
                     gene prediction method: BestRefSeq."
                     /note="color: #a6acb3; direction: RIGHT"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /transcript_id="NM_001289746.2"
     mRNA            256..270
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase,
                     transcript variant 3"
                     /label=GAPDH mRNA
                     /note="Derived by automated computational analysis using
                     gene prediction method: BestRefSeq."
                     /note="color: #a6acb3; direction: RIGHT"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /transcript_id="NM_001289745.3"
     mRNA            256..270
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase,
                     transcript variant 1"
                     /label=GAPDH mRNA
                     /note="Derived by automated computational analysis using
                     gene prediction method: BestRefSeq."
                     /note="color: #a6acb3; direction: RIGHT"
                     /db_xref="Ensembl:ENST00000229239.10"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /transcript_id="NM_002046.7"
     misc_RNA        256..270
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase,
                     transcript variant 6"
                     /note="Derived by automated computational analysis using
                     gene prediction method: BestRefSeq."
                     /note="color: #a6acb3; direction: RIGHT"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /transcript_id="NR_152150.2"
     mRNA            256..270
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase,
                     transcript variant 7"
                     /label=GAPDH mRNA
                     /note="Derived by automated computational analysis using
                     gene prediction method: BestRefSeq."
                     /note="color: #a6acb3; direction: RIGHT"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /transcript_id="NM_001357943.2"
     CDS             256..270
                     /codon_start=1
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase isoform
                     1"
                     /label=GAPDH
                     /note="isoform 1 is encoded by transcript variant 3;
                     Derived by automated computational analysis using gene 
                     prediction method: BestRefSeq."
                     /note="color: #993366"
                     /db_xref="CCDS:CCDS8549.1"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /protein_id="NP_001276674.1"
                     /translation="YDNE"
     CDS             256..270
                     /codon_start=1
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase isoform
                     1"
                     /label=GAPDH
                     /note="isoform 1 is encoded by transcript variant 4;
                     Derived by automated computational analysis using gene 
                     prediction method: BestRefSeq."
                     /note="color: #993366"
                     /db_xref="CCDS:CCDS8549.1"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /protein_id="NP_001276675.1"
                     /translation="YDNE"
     CDS             256..270
                     /codon_start=1
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase isoform
                     1"
                     /label=GAPDH
                     /note="isoform 1 is encoded by transcript variant 1;
                     Derived by automated computational analysis using gene 
                     prediction method: BestRefSeq."
                     /note="color: #993366"
                     /db_xref="CCDS:CCDS8549.1"
                     /db_xref="Ensembl:ENSP00000229239.5"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /protein_id="NP_002037.2"
                     /translation="YDNE"
     CDS             256..270
                     /codon_start=1
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase isoform
                     4"
                     /label=GAPDH
                     /note="isoform 4 is encoded by transcript variant 7;
                     Derived by automated computational analysis using gene 
                     prediction method: BestRefSeq."
                     /note="color: #993366"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /protein_id="NP_001344872.1"
                     /translation="YDNE"
     mRNA            256..270
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase,
                     transcript variant 2"
                     /label=GAPDH mRNA
                     /note="Derived by automated computational analysis using
                     gene prediction method: BestRefSeq."
                     /note="color: #a6acb3; direction: RIGHT"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /transcript_id="NM_001256799.3"
     CDS             256..270
                     /codon_start=1
                     /gene="GAPDH"
                     /gene_synonym="G3PD; GAPD; HEL-S-162eP"
                     /product="glyceraldehyde-3-phosphate dehydrogenase isoform
                     2"
                     /label=GAPDH
                     /note="isoform 2 is encoded by transcript variant 2;
                     Derived by automated computational analysis using gene 
                     prediction method: BestRefSeq."
                     /note="color: #993366"
                     /db_xref="CCDS:CCDS58201.1"
                     /db_xref="GeneID:2597"
                     /db_xref="HGNC:HGNC:4141"
                     /db_xref="MIM:138400"
                     /protein_id="NP_001243728.1"
                     /translation="YDNE"
     misc_feature    268..270
                     /label=3'arm
                     /note="color: #ff6600"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     primer_bind     complement(322..341)
                     /label=Ubi-Lib R
                     /note="color: black; sequence: gactcggtgccactttttca; added:
                     2025-04-28"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
                     /note="color: #ffffff"
     intron          949..1176
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
                     /note="color: #------"
     regulatory      1188..1197
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             1197..1262
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /note="color: #cc99b2
                     Cleavage site after base 1262"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1269..1289
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     CDS             1314..5414
                     /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"
                     /note="color: #993366"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     complement(4917..4937)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
                     /note="color: black; sequence: CGTCGCCGTCCAGCTCGACCAG"
     CDS             5415..5462
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /note="color: #993366"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5478..5531
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /note="color: #cc99b2
                     Cleavage site after base 5528"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(5532..5534,5535..6245)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="This feature has 2 segments:
                        1: 5532 .. 5534 / #05fd14 / 1a
                        2: 5535 .. 6245 / #05fd14"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5532..5534,5535..6245)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 2 segments:
                        1: 5532 .. 5534 / #05fc14 / 1a
                        2: 5535 .. 6245 / #05fc14"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5574..5595)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
                     /note="color: black; sequence: CGTCGCCGTCCAGCTCGACCAG"
     primer_bind     complement(5835..5854)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
                     /note="color: black; sequence: GTCTTGTAGTTGCCGTCGTC"
     primer_bind     6182..6203
                     /label=EGFP-C
                     /note="EGFP, forward primer"
                     /note="color: black; sequence: CATGGTCCTGCTGGAGTTCGTG"
     primer_bind     complement(6273..6290)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
                     /note="color: black; sequence: TAGAAGGCACAGTCGAGG"
     polyA_signal    6279..6486
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     repeat_region   6495..6635
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     repeat_region   6495..6624
                     /label=AAV2 ITR
                     /note="color: #ffcc99"
     rep_origin      6710..7165
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     primer_bind     complement(6797..6816)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="color: black; sequence: AGGGAAGAAAGCGAAAGGAG"
     primer_bind     7007..7028
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="color: black; sequence: GTGGACTCTTGTTCCAAACTGG"
     primer_bind     complement(7182..7201)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /note="color: black; sequence: CGGCATCAGAGCAGATTGTA"
     primer_bind     7301..7323
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
                     /note="color: black; sequence: CCGGGAGCTGCATGTGTCAGAGG"
     primer_bind     complement(7361..7379)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
                     /note="color: black; sequence: AATAGGCGTATCACGAGGC"
     promoter        7447..7551
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             join(7552..7620,7621..8412)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 7552 .. 7620 / #ccffcc / signal sequence
                        2: 7621 .. 8412 / #ccffcc
                     Cleavage site after base 7620"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7770..7789)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: black; sequence: ATAATACCGCGCCACATAGC"
     rep_origin      8583..9171
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     9072..9091
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg tctaggtatg acaacgaatt gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 ggggggggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
      901 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
      961 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
     1021 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     1081 tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat
     1141 tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact
     1201 ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata
     1261 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga
     1321 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg
     1381 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1441 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1501 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1561 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1621 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1681 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1741 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1801 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1861 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1921 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     1981 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     2041 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     2101 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     2161 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     2221 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     2281 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     2341 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     2401 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2461 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2521 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2581 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2641 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2701 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2761 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2821 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2881 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     2941 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     3001 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     3061 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     3121 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     3181 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     3241 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     3301 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     3361 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     3421 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3481 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3541 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3601 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3661 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3721 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3781 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     3841 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3901 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     3961 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     4021 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     4081 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     4141 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     4201 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     4261 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     4321 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     4381 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4441 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4501 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4561 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4621 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4681 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4741 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4801 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4861 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4921 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     4981 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     5041 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     5101 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     5161 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     5221 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     5281 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     5341 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     5401 agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa
     5461 aggaattcgg cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga
     5521 atcctggccc agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     5581 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     5641 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     5701 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     5761 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     5821 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     5881 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     5941 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     6001 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     6061 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     6121 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     6181 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     6241 acaaggaatt ctaactagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     6301 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     6361 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     6421 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
     6481 tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     6541 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     6601 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     6661 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     6721 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     6781 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     6841 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     6901 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     6961 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     7021 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     7081 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     7141 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     7201 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     7261 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     7321 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     7381 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     7441 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     7501 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     7561 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     7621 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     7681 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     7741 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     7801 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     7861 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     7921 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     7981 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     8041 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     8101 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     8161 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     8221 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     8281 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     8341 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     8401 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     8461 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     8521 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     8581 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     8641 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     8701 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     8761 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     8821 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     8881 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     8941 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     9001 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     9061 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     9121 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     9181 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
