LOCUS       sequence-339220-        9274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 with 5' gRNA for paired CRISPR KO of miR-290-295 cluster.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9274)
  AUTHORS   Schaefer M, Nabih A, Spies D, Hermes V, Bodak M, Wischnewski H, 
            Stalder P, Ngondo RP, Liechti LA, Sajic T, Aebersold R, Gatfield D, 
            Ciaudo C
  TITLE     Global and precise identification of functional miRNA targets in 
            mESCs by integrative analysis.
  JOURNAL   EMBO Rep. 2022 Jul 28:e54762. doi: 10.15252/embr.202254762.
  PUBMED    35899551
REFERENCE   2  (bases 1 to 9274)
  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"; journalName: "EMBO Rep. 
            2022 Jul 28:e54762. doi: 10.15252/embr.202254762."
COMMENT     Sequence Label: pX458_miR-290-295KO-gRNA1
FEATURES             Location/Qualifiers
     source          1..9274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          991..1218
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1230..1239
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1239..1304
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1311..1331
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1356..5456
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1356..5456
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5457..5504
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5520..5573
                     /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."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(5574..5576,5577..6287)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5574..5576,5577..6287)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5877..5896)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6224..6245
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6315..6332)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6321..6528
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6537..6677
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6537..6666
                     /label=AAV2 ITR
     rep_origin      6752..7207
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6839..6858)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7049..7070
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7224..7243)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7343..7365
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7403..7421)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7489..7593
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7594..7662,7663..8454)
                     /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     complement(7812..7831)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8625..9213
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9114..9133
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
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 tagtacatcg gtctaactca 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 gggggggggg gggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca
      901 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg
      961 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg
     1021 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
     1081 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag caagaggtaa
     1141 gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga gcacctgcct
     1201 gaaatcactt tttttcaggt tggaccggtg ccaccatgga ctataaggac cacgacggag
     1261 actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga
     1321 agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg
     1381 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
     1441 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
     1501 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
     1561 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
     1621 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
     1681 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
     1741 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     1801 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     1861 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
     1921 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     1981 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     2041 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc
     2101 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     2161 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     2221 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     2281 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     2341 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     2401 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
     2461 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     2521 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     2581 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     2641 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     2701 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     2761 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
     2821 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     2881 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
     2941 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
     3001 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     3061 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct
     3121 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg
     3181 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag
     3241 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc
     3301 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca
     3361 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca
     3421 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga
     3481 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca
     3541 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag
     3601 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca
     3661 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag
     3721 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga
     3781 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca
     3841 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg
     3901 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg
     3961 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca
     4021 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg
     4081 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc
     4141 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga
     4201 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt
     4261 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga
     4321 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt
     4381 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg
     4441 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga
     4501 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg
     4561 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc
     4621 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta
     4681 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga
     4741 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg
     4801 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg
     4861 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag
     4921 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc
     4981 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct
     5041 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg
     5101 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca
     5161 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag
     5221 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca
     5281 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca
     5341 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc
     5401 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa
     5461 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaaggaattc ggcagtggag
     5521 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccagtgagca
     5581 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     5641 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     5701 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     5761 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     5821 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     5881 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     5941 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     6001 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     6061 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     6121 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     6181 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     6241 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggaa ttctaactag
     6301 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     6361 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     6421 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     6481 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga
     6541 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     6601 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     6661 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     6721 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     6781 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     6841 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     6901 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     6961 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     7021 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     7081 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     7141 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     7201 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     7261 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     7321 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     7381 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     7441 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     7501 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     7561 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     7621 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     7681 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     7741 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     7801 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     7861 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     7921 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     7981 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     8041 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     8101 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     8161 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     8221 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     8281 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     8341 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     8401 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     8461 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     8521 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     8581 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     8641 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     8701 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     8761 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     8821 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     8881 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     8941 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     9001 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     9061 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     9121 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     9181 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     9241 ttcctggcct tttgctggcc ttttgctcac atgt
//
