LOCUS       sequence-485643-       11989 bp    DNA     circular SYN 09-SEP-2025
DEFINITION  CRISPR-Cas9 genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11989)
  AUTHORS   Hossain MJ, O'Connor TJ
  TITLE     An efficient and cost-effective method for disrupting genes in 
            RAW264.7 macrophages using CRISPR-Cas9.
  JOURNAL   PLoS One. 2024 Mar 14;19(3):e0299513. doi: 
            10.1371/journal.pone.0299513. eCollection 2024.
  PUBMED    38483963
REFERENCE   2  (bases 1 to 11989)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 9, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2024-03-14"; volume: "19(3):e0299513. doi"; pages: " 
            10.1371/journal.pone.0299513. eCollection 2024"
COMMENT     Sequence Label: pCRISPR-EASY
FEATURES             Location/Qualifiers
     source          1..11989
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        173..421
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     173..193
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     344..363
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1163..1238
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1349..1368)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1656..2834
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1887..2825
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2782..2802
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     2851..2870
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2851..2869
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2925..2934
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2937..2957
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2958..2978
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2979..2999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3003..3005,3006..3716)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3045..3066)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3306..3325)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3653..3674
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3726..3782
                     /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"
     regulatory      3804..3813
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3810..7913
                     /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             3810..7913
                     /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             7926..7946
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7947..7967
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7968..7988
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7989..8012
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(8078..8095)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8084..8308
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8354..8782
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8441..8460)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8651..8672
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8791..8811)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8796..9125
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8976..9111
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9038..9057
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9173..9220
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9239..9637
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    9795..9916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9832..9851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9886..9905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9965..9981)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9965..9981)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9978..10000)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9989..10005
                     /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(10013..10019,10020..10037,10038..10043))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10058..10079
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10196..10213)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10356..10944)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10436..10455)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11115..11906,11907..11975))
                     /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     11738..11757
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(11976..11989,1..91))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
       61 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
      121 tccgggttta ttacagggac agcagagatc cagtttggtt aattaaggta ccgagggcct
      181 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattag
      241 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
      301 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
      361 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
      421 cggagacggt tgtaaatgag cacacaaaat acacatgcta aaatattata ttctatgacc
      481 tttataaaat caaccaaaat cttcttttta ataactttag tatcaataat tagaattttt
      541 atgttccttt ttgcaaactt ttaataaaaa tgagcaaaat aaaaaaacgc tagttttagt
      601 aactcgcgtt gttttcttca cctttaataa tagctactcc accacttgtt cctaagcggt
      661 cagctcctgc ttcaatcatt ttttgagcat cttcaaatgt tctaactcca ccagctgctg
      721 agcgattcta tcacccaaat cacttgttac aaaacttgaa tatagagccg gattttttgg
      781 aatgctattt aagcgtttta gatttgataa ttttcaatca attaaaattg gtcctgtttg
      841 atttccatct aatcttacaa aatgaccatc atgttttatt gccattccaa atctgtcagc
      901 atctgggtca ttcataataa taatatctgc atcatgttta ataccatatt caagcggtat
      961 ttttcatgca ggatcaaatt ctggatttgg atttacaaca tttttaaatg tttcatcttc
     1021 aaatgcatgc tcttcaacct caataacgtt atatcctgat tcacgtaata tttttggggt
     1081 aaatttagtt cctgttccat taactgcgct aaaaataatt tttaaatctt ttttagcttc
     1141 ttgctctttt ttgtacgtct ctgttttaga gctagaaata gcaagttaaa ataaggctag
     1201 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgaattc gctagctagg
     1261 tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg acgtcgacgg atcgggagat
     1321 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     1381 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     1441 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     1501 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactaggc
     1561 ttttgcaaaa agctttgcaa agatggataa agttttaaac agagaggaat ctttgcagct
     1621 aatggacctt ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag
     1681 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg
     1741 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
     1801 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
     1861 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc
     1921 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga
     1981 ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag
     2041 gagccccttc gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc
     2101 gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa
     2161 atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc
     2221 aagatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt
     2281 cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg
     2341 ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc
     2401 cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc
     2461 ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg
     2521 gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact
     2581 ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt
     2641 cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg
     2701 agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg
     2761 agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat
     2821 ttcaggtgtc gtgaggaatt agcttggtac taatacgact cactataggg agacccaagc
     2881 tggctaggta agcttggtac cgagctcgga tccactagtc ccgggccacc atggtgccta
     2941 agaagaagag gaaggttcct aagaagaaga ggaaggttcc taagaagaag aggaaggtta
     3001 acgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     3061 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     3121 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     3181 tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc
     3241 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     3301 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     3361 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     3421 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     3481 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     3541 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     3601 acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     3661 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggat
     3721 ccggagccac gaacttctct ctgttaaagc aagcaggcga cgtggaagaa aaccccggtc
     3781 ctggtctaga ggatcgaacc cttgccacca tggacaagaa gtactccatt gggctcgata
     3841 tcggcacaaa cagcgtcggc tgggccgtca ttacggacga gtacaaggtg ccgagcaaaa
     3901 aattcaaagt tctgggcaat accgatcgcc acagcataaa gaagaacctc attggcgccc
     3961 tcctgttcga ctccggggaa acggccgaag ccacgcggct caaaagaaca gcacggcgca
     4021 gatatacccg cagaaagaat cggatctgct acctgcagga gatctttagt aatgagatgg
     4081 ctaaggtgga tgactctttc ttccataggc tggaggagtc ctttttggtg gaggaggata
     4141 aaaagcacga gcgccaccca atctttggca atatcgtgga cgaggtggcg taccatgaaa
     4201 agtacccaac catatatcat ctgaggaaga agcttgtaga cagtactgat aaggctgact
     4261 tgcggttgat ctatctcgcg ctggcgcata tgatcaaatt tcggggacac ttcctcatcg
     4321 agggggacct gaacccagac aacagcgatg tcgacaaact ctttatccaa ctggttcaga
     4381 cttacaatca gcttttcgaa gagaacccga tcaacgcatc cggagttgac gccaaagcaa
     4441 tcctgagcgc taggctgtcc aaatcccggc ggctcgaaaa cctcatcgca cagctccctg
     4501 gggagaagaa gaacggcctg tttggtaatc ttatcgccct gtcactcggg ctgaccccca
     4561 actttaaatc taacttcgac ctggccgaag atgccaagct tcaactgagc aaagacacct
     4621 acgatgatga tctcgacaat ctgctggccc agatcggcga ccagtacgca gacctttttt
     4681 tggcggcaaa gaacctgtca gacgccattc tgctgagtga tattctgcga gtgaacacgg
     4741 agatcaccaa agctccgctg agcgctagta tgatcaagcg ctatgatgag caccaccaag
     4801 acttgacttt gctgaaggcc cttgtcagac agcaactgcc tgagaagtac aaggaaattt
     4861 tcttcgatca gtctaaaaat ggctacgccg gatacattga cggcggagca agccaggagg
     4921 aattttacaa atttattaag cccatcttgg aaaaaatgga cggcaccgag gagctgctgg
     4981 taaagcttaa cagagaagat ctgttgcgca aacagcgcac tttcgacaat ggaagcatcc
     5041 cccaccagat tcacctgggc gaactgcacg ctatcctcag gcggcaagag gatttctacc
     5101 cctttttgaa agataacagg gaaaagattg agaaaatcct cacatttcgg ataccctact
     5161 atgtaggccc cctcgcccgg ggaaattcca gattcgcgtg gatgactcgc aaatcagaag
     5221 agaccatcac tccctggaac ttcgaggaag tcgtggataa gggggcctct gcccagtcct
     5281 tcatcgaaag gatgactaac tttgataaaa atctgcctaa cgaaaaggtg cttcctaaac
     5341 actctctgct gtacgagtac ttcacagttt ataacgagct caccaaggtc aaatacgtca
     5401 cagaagggat gagaaagcca gcattcctgt ctggagagca gaagaaagct atcgtggacc
     5461 tcctcttcaa gacgaaccgg aaagttaccg tgaaacagct caaagaagac tatttcaaaa
     5521 agattgaatg tttcgactct gttgaaatca gcggagtgga ggatcgcttc aacgcatccc
     5581 tgggaacgta tcacgatctc ctgaaaatca ttaaagacaa ggacttcctg gacaatgagg
     5641 agaacgagga cattcttgag gacattgtcc tcacccttac gttgtttgaa gatagggaga
     5701 tgattgaaga acgcttgaaa acttacgctc atctcttcga cgacaaagtc atgaaacagc
     5761 tcaagaggcg ccgatataca ggatgggggc ggctgtcaag aaaactgatc aatgggatcc
     5821 gagacaagca gagtggaaag acaatcctgg attttcttaa gtccgatgga tttgccaacc
     5881 ggaacttcat gcagttgatc catgatgact ctctcacctt taaggaggac atccagaaag
     5941 cacaagtttc tggccagggg gacagtcttc acgagcacat cgctaatctt gcaggtagcc
     6001 cagctatcaa aaagggaata ctgcagaccg ttaaggtcgt ggatgaactc gtcaaagtaa
     6061 tgggaaggca taagcccgag aatatcgtta tcgagatggc ccgagagaac caaactaccc
     6121 agaagggaca gaagaacagt agggaaagga tgaagaggat tgaagagggt ataaaagaac
     6181 tggggtccca aatccttaag gaacacccag ttgaaaacac ccagcttcag aatgagaagc
     6241 tctacctgta ctacctgcag aacggcaggg acatgtacgt ggatcaggaa ctggacatca
     6301 atcggctctc cgactacgac gtggatcata tcgtgcccca gtcttttctc aaagatgatt
     6361 ctattgataa taaagtgttg acaagatccg ataaaaatag agggaagagt gataacgtcc
     6421 cctcagaaga agttgtcaag aaaatgaaaa attattggcg gcagctgctg aacgccaaac
     6481 tgatcacaca acggaagttc gataatctga ctaaggctga acgaggtggc ctgtctgagt
     6541 tggataaagc cggcttcatc aaaaggcagc ttgttgagac acgccagatc accaagcacg
     6601 tggcccaaat tctcgattca cgcatgaaca ccaagtacga tgaaaatgac aaactgattc
     6661 gagaggtgaa agttattact ctgaagtcta agctggtctc agatttcaga aaggactttc
     6721 agttttataa ggtgagagag atcaacaatt accaccatgc gcatgatgcc tacctgaatg
     6781 cagtggtagg cactgcactt atcaaaaaat atcccaagct tgaatctgaa tttgtttacg
     6841 gagactataa agtgtacgat gttaggaaaa tgatcgcaaa gtctgagcag gaaataggca
     6901 aggccaccgc taagtacttc ttttacagca atattatgaa ttttttcaag accgagatta
     6961 cactggccaa tggagagatt cggaagcgac cacttatcga aacaaacgga gaaacaggag
     7021 aaatcgtgtg ggacaagggt agggatttcg cgacagtccg gaaggtcctg tccatgccgc
     7081 aggtgaacat cgttaaaaag accgaagtac agaccggagg cttctccaag gaaagtatcc
     7141 tcccgaaaag gaacagcgac aagctgatcg cacgcaaaaa agattgggac cccaagaaat
     7201 acggcggatt cgattctcct acagtcgctt acagtgtact ggttgtggcc aaagtggaga
     7261 aagggaagtc taaaaaactc aaaagcgtca aggaactgct gggcatcaca atcatggagc
     7321 gatcaagctt cgaaaaaaac cccatcgact ttctcgaggc gaaaggatat aaagaggtca
     7381 aaaaagacct catcattaag cttcccaagt actctctctt tgagcttgaa aacggccgga
     7441 aacgaatgct cgctagtgcg ggcgagctgc agaaaggtaa cgagctggca ctgccctcta
     7501 aatacgttaa tttcttgtat ctggccagcc actatgaaaa gctcaaaggg tctcccgaag
     7561 ataatgagca gaagcagctg ttcgtggaac aacacaaaca ctaccttgat gagatcatcg
     7621 agcaaataag cgaattctcc aaaagagtga tcctcgccga cgctaacctc gataaggtgc
     7681 tttctgctta caataagcac agggataagc ccatcaggga gcaggcagaa aacattatcc
     7741 acttgtttac tctgaccaac ttgggcgcgc ctgccgcctt caagtacttc gacaccacca
     7801 tcgacagaaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     7861 gcatcaccgg cctgtacgag acaagaatcg acctgagcca gctgggcggc gacagcagag
     7921 ccgaccctaa gaaaaagcgg aaggtgccca aaaagaaacg caaggtgcca aagaaaaaaa
     7981 gaaaggtgga ctacaaggac gacgacgaca agtgaaaggg ttcgatccct accggttagt
     8041 aatgagttta tcatcgatgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     8101 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     8161 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     8221 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     8281 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     8341 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     8401 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     8461 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     8521 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     8581 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     8641 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     8701 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     8761 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     8821 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     8881 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     8941 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     9001 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     9061 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     9121 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa
     9181 ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat
     9241 ggccaagcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa
     9301 cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg
     9361 catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt
     9421 gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa
     9481 tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca
     9541 tcctgggatc aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg
     9601 tgaattgctg ccctctggtt atgtgtggga gggctaagca cttcgtggcc gaggagcagg
     9661 actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     9721 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     9781 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     9841 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     9901 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     9961 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
    10021 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
    10081 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
    10141 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
    10201 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
    10261 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
    10321 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
    10381 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
    10441 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
    10501 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
    10561 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
    10621 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
    10681 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
    10741 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
    10801 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
    10861 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
    10921 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
    10981 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
    11041 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
    11101 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
    11161 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
    11221 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
    11281 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
    11341 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
    11401 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
    11461 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
    11521 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
    11581 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
    11641 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
    11701 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
    11761 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
    11821 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
    11881 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
    11941 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttc
//
