LOCUS       sequence-191220-       16581 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  To episomally express codon optimized Cas9 and chimeric guide RNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 16581)
  AUTHORS   Li L, Gao F, Wu S
  TITLE     An episomal CRISPR/Cas9 system to derive vector-free gene modified 
            mammalian cells.
  JOURNAL   Protein Cell. 2016 Sep;7(9):689-91. doi: 10.1007/s13238-016-0299-9.
  PUBMED    27472953
REFERENCE   2  (bases 1 to 16581)
  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"; journalName: 
            "Protein Cell"; date: "2016-09"; volume: "7"; issue: "9"; pages: 
            "689-91"
COMMENT     Sequence Label: pCRISPR-S12
FEATURES             Location/Qualifiers
     source          1..16581
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(823..841)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        909..1013
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1014..1082,1083..1874)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1232..1251)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2045..2633
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2534..2553
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3064..3209
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     CDS             3252..4289
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     polyA_signal    4331..4379
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     enhancer        4962..5341
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5342..5545
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5496..5516
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          5637..5769
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      5799..5808
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5808..5873
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5880..5900
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5925..10025
                     /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             5925..10025
                     /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             10026..10073
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10083..10148
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(10149..10151,10152..10154,10155..10868)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(10149..10151,10152..10154,10155..10865)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(10455..10474)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     10802..10823
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(10893..10910)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10899..11106
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        11161..11409
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     11161..11181
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     11332..11351
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        11431..11506
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        11611..11682
                     /gene="bla"
                     /label=AmpR promoter
     promoter        11684..12041
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        11684..11875
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(11707..11727)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      11892..12027
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             12094..12693
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(12094..12113)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     12590..12610
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    12932..12979
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      13449..15238
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
ORIGIN
        1 cccctcctcc tgctcctgcc cctcctgccc ctcctgcccc tcctgcccct cctcctgctc
       61 ctgcccctcc tcctgctcct gcccctcctg ctcctgcccc tcccgctcct gctcctgctc
      121 ctgttccacc gtgggtccct ttgcagccaa tgcaacttgg acgtttttgg ggtctccgga
      181 caccatctct atgtcttggc cctgatcctg agccgcccgg ggctcctggt cttccgcctc
      241 ctcgtcctcg tcctcttccc cgtcctcgtc catggttatc accccctctt ctttgaggtc
      301 cactgccgcc ggagccttct ggtccagatg tgtctccctt ctctcctagg ccatttccag
      361 gtcctgtacc tggcccctcg tcagacatga ttcacactaa aagagatcaa tagacatctt
      421 tattagacga cgctcagtga atacagggag tgcagactcc tgccccctcc aacagccccc
      481 ccaccctcat ccccttcatg gtcgctgtca gacagatcca ggtctgaaaa ttccccatcc
      541 tccgaaccat cctcgtcctc atcaccaatt actcgcagcc cggaaaactc ccgctgaaca
      601 tcctcaagat ttgcgtcctg agcctcaagc caggcctcaa attcctcgtc cccctttttg
      661 ctggacggta gggatgggga ttctcgggac ccctcctctt cctcttcaag gtcaccagac
      721 agagatgcta ctggggcaac ggaagaaaag ctgggtgcgg cctgtgagga tcagcttatc
      781 gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat
      841 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
      901 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      961 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1021 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     1081 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1141 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1201 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     1261 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1321 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1381 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1441 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1501 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     1561 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1621 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1681 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1741 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1801 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1861 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1921 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1981 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2041 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2101 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2161 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     2221 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2281 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2341 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2401 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2461 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2521 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2581 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2641 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttgaagctgt ccctgatggt
     2701 cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag
     2761 cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt
     2821 agcccagcgc gtcggccccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat
     2881 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc
     2941 tccaattctt ggagtggtga atccgttagc gaggtgccgc cctgcttcat ccccgtggcc
     3001 cgttgctcgc gtttgctggc ggtgtccccg gaagaaatat atttgcatgt ctttagttct
     3061 atgatgacac aaaccccgcc cagcgtcttg tcattggcga attcgaacac gcagatgcag
     3121 tcggggcggc gcggtccgag gtccacttcg catattaagg tgacgcgtgt ggcctcgaac
     3181 accgagcgac cctgcagcga cccgcttaac agcgtcaaca gcgtgccgca gatcccgggg
     3241 ggcaatgaga tatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     3301 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     3361 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     3421 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     3481 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     3541 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     3601 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     3661 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     3721 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     3781 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     3841 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     3901 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     3961 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     4021 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     4081 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     4141 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     4201 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     4261 cggatcggga gatgggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     4321 cgcgctatga cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttca
     4381 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     4441 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     4501 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagccactg gccccgtggg
     4561 ttagggacgg ggtcccccat ggggaatggt ttatggttcg tgggggttat tattttgggc
     4621 gttgcgtggg gtcaggtcca cgactggact gagcagacag acccatggtt tttggatggc
     4681 ctgggcatgg accgcatgta ctggcgcgac acgaacaccg ggcgtctgtg gctgccaaac
     4741 acccccgacc cccaaaaacc accgcgcgga tttctggcgt gccaagctag tcgattaatt
     4801 aatagggata acagggtaat tttgtcaata ttggccatta gccatattat tcattggtta
     4861 tatagcataa atcaatattg gctattggcc attgcatacg ttgtatctat atcataatat
     4921 gtacatttat attggctcat gtccaatatg accgccatgt tggcattgat tattgactag
     4981 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     5041 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     5101 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     5161 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     5221 tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     5281 gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     5341 ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt
     5401 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     5461 ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg
     5521 gtgggaggtc tatataagca gaggtcgttt agtgaaccgt cagatcacta gtagctttat
     5581 tgcggtagtt tatcacagtt aaattgctaa cgcagtcagt gctcgactga tcacaggtaa
     5641 gtatcaaggt tacaagacag gtttaaggag gccaatagaa actgggcttg tcgagacaga
     5701 gaagattctt gcgtttctga taggcaccta ttggtcttac tgacatccac tttgcctttc
     5761 tctccacagg ggtaccgaag ccgctagtcg acaccggtgc caccatggac tataaggacc
     5821 acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc
     5881 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca
     5941 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     6001 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     6061 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     6121 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     6181 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     6241 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     6301 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     6361 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     6421 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     6481 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     6541 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     6601 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     6661 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     6721 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     6781 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     6841 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     6901 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     6961 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     7021 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     7081 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     7141 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     7201 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     7261 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     7321 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     7381 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     7441 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     7501 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     7561 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     7621 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     7681 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     7741 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     7801 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     7861 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     7921 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     7981 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     8041 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     8101 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     8161 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     8221 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     8281 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     8341 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     8401 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     8461 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     8521 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     8581 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     8641 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     8701 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     8761 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     8821 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     8881 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     8941 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     9001 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     9061 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     9121 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     9181 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     9241 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     9301 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     9361 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     9421 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     9481 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     9541 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     9601 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     9661 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     9721 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     9781 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     9841 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     9901 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     9961 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
    10021 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagggaagtg
    10081 gcgtgaaaca gactttgaat tttgaccttc tcaagttggc gggagacgtg gagtccaacc
    10141 cagggcccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
    10201 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
    10261 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
    10321 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
    10381 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
    10441 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
    10501 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
    10561 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
    10621 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
    10681 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
    10741 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
    10801 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
    10861 acaagtgaga attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
    10921 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
    10981 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
    11041 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
    11101 tggggagcgg ccgcgccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
    11161 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
    11221 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
    11281 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
    11341 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
    11401 cgaaacaccg attttgtttt gatgtattca gttttagagc tagaaatagc aagttaaaat
    11461 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
    11521 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcac caattctgca
    11581 gacaaatggc ggatcctatt tttctaaata cattcaaata tgtatccgct catgagacaa
    11641 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
    11701 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
    11761 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
    11821 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
    11881 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
    11941 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
    12001 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
    12061 tgaggatcgt ttcgctctag agtggccaca accatgaccg agtacaagcc cacggtgcgc
    12121 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
    12181 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
    12241 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
    12301 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
    12361 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
    12421 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
    12481 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
    12541 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
    12601 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
    12661 acctggtgca tgacccgcaa gcccggtgcc tgagataaac gcgggactct ggggttcgaa
    12721 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
    12781 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
    12841 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
    12901 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
    12961 ggtgttgggt cgtttgttcg gcgcgcctcg actctagagg atcgatcccc gccccggacg
    13021 aactaaacct gactacgaca tctctgcccc ttcttcgcgg ggcagtgcat gtaatccctt
    13081 cagttggttg gtacaacttg ccaactgggc cctgttccac atgtgacacg gggggggacc
    13141 aaacacaaag gggttctctg actgtagttg acatccttat aaatggatgt gcacatttgc
    13201 caacactgag tggctttcat cctggagcag actttgcagt ctgtggactg caacacaaca
    13261 ttgcctttat gtgtaactct tggctgaagc tcttacacca atgctggggg acatgtacct
    13321 cccaggggcc caggaagact acgggaggct acaccaacgt caatcagagg ggcctgtgta
    13381 gctaccgata agcggaccct caagagggca ttagcaatag tgtttataag gcccccttgt
    13441 taaccctaaa cgggtagcat atgcttcccg ggtagtagta tatactatcc agactaaccc
    13501 taattcaata gcatatgtta cccaacggga agcatatgct atcgaattag ggttagtaaa
    13561 agggtcctaa ggaacagcga tatctcccac cccatgagct gtcacggttt tatttacatg
    13621 gggtcaggat tccacgaggg tagtgaacca ttttagtcac aagggcagtg gctgaagatc
    13681 aaggagcggg cagtgaactc tcctgaatct tcgcctgctt cttcattctc cttcgtttag
    13741 ctaatagaat aactgctgag ttgtgaacag taaggtgtat gtgaggtgct cgaaaacaag
    13801 gtttcaggtg acgcccccag aataaaattt ggacgggggg ttcagtggtg gcattgtgct
    13861 atgacaccaa tataaccctc acaaacccct tgggcaataa atactagtgt aggaatgaaa
    13921 cattctgaat atctttaaca atagaaatcc atggggtggg gacaagccgt aaagactgga
    13981 tgtccatctc acacgaattt atggctatgg gcaacacata atcctagtgc aatatgatac
    14041 tggggttatt aagatgtgtc ccaggcaggg accaagacag gtgaaccatg ttgttacact
    14101 ctatttgtaa caaggggaaa gagagtggac gccgacagca gcggactcca ctggttgtct
    14161 ctaacacccc cgaaaattaa acggggctcc acgccaatgg ggcccataaa caaagacaag
    14221 tggccactct tttttttgaa attgtggagt gggggcacgc gtcagccccc acacgccgcc
    14281 ctgcggtttt ggactgtaaa ataagggtgt aataacttgg ctgattgtaa ccccgctaac
    14341 cactgcggtc aaaccacttg cccacaaaac cactaatggc accccgggga atacctgcat
    14401 aagtaggtgg gcgggccaag ataggggcgc gattgctgcg atctggagga caaattacac
    14461 acacttgcgc ctgagcgcca agcacagggt tgttggtcct catattcacg aggtcgctga
    14521 gagcacggtg ggctaatgtt gccatgggta gcatatacta cccaaatatc tggatagcat
    14581 atgctatcct aatctatatc tgggtagcat aggctatcct aatctatatc tgggtagcat
    14641 atgctatcct aatctatatc tgggtagtat atgctatcct aatttatatc tgggtagcat
    14701 aggctatcct aatctatatc tgggtagcat atgctatcct aatctatatc tgggtagtat
    14761 atgctatcct aatctgtatc cgggtagcat atgctatcct aatagagatt agggtagtat
    14821 atgctatcct aatttatatc tgggtagcat atactaccca aatatctgga tagcatatgc
    14881 tatcctaatc tatatctggg tagcatatgc tatcctaatc tatatctggg tagcataggc
    14941 tatcctaatc tatatctggg tagcatatgc tatcctaatc tatatctggg tagtatatgc
    15001 tatcctaatt tatatctggg tagcataggc tatcctaatc tatatctggg tagcatatgc
    15061 tatcctaatc tatatctggg tagtatatgc tatcctaatc tgtatccggg tagcatatgc
    15121 tatcctcatg catatacagt cagcatatga tacccagtag tagagtggga gtgctatcct
    15181 ttgcatatgc cgccacctcc caagggggcg tgaattttcg ctgcttgtcc ttttcctgct
    15241 ggttgctccc attcttaggt gaatttaagg aggccaggct aaagccgtcg catgtctgat
    15301 tgctcaccag gtaaatgtcg ctaatgtttt ccaacgcgag aaggtgttga gcgcggagct
    15361 gagtgacgtg acaacatggg tatgcccaat tgccccatgt tgggaggacg aaaatggtga
    15421 caagacagat ggccagaaat acaccaacag cacgcatgat gtctactggg gatttattct
    15481 ttagtgcggg ggaatacacg gcttttaata cgattgaggg cgtctcctaa caagttacat
    15541 cactcctgcc cttcctcacc ctcatctcca tcacctcctt catctccgtc atctccgtca
    15601 tcaccctccg cggcagcccc ttccaccata ggtggaaacc agggaggcaa atctactcca
    15661 tcgtcaaagc tgcacacagt caccctgata ttgcaggtag gagcgggctt tgtcataaca
    15721 aggtccttaa tcgcatcctt caaaacctca gcaaatatat gagtttgtaa aaagaccatg
    15781 aaataacaga caatggactc ccttagcggg ccaggttgtg ggccgggtcc aggggccatt
    15841 ccaaagggga gacgactcaa tggtgtaaga cgacattgtg gaatagcaag ggcagttcct
    15901 cgccttaggt tgtaaaggga ggtcttacta cctccatata cgaacacacc ggcgacccaa
    15961 gttccttcgt cggtagtcct ttctacgtga ctcctagcca ggagagctct taaaccttct
    16021 gcaatgttct caaatttcgg gttggaacct ccttgaccac gatgctttcc aaaccaccct
    16081 ccttttttgc gcctgcctcc atcaccctga ccccggggtc cagtgcttgg gccttctcct
    16141 gggtcatctg cggggccctg ctctatcgct cccgggggca cgtcaggctc accatctggg
    16201 ccaccttctt ggtggtattc aaaataatcg gcttccccta cagggtggaa aaatggcctt
    16261 ctacctggag ggggcctgcg cggtggagac ccggatgatg atgactgact actgggactc
    16321 ctgggcctct tttctccacg tccacgacct ctccccctgg ctctttcacg acttcccccc
    16381 ctggctcttt cacgtcctct accccggcgg cctccactac ctcctcgacc ccggcctcca
    16441 ctacctcctc gaccccggcc tccactgcct cctcgacccc ggcctccacc tcctgctcct
    16501 gcccctcctg ctcctgcccc tcctcctgct cctgcccctc ctgcccctcc tgctcctgcc
    16561 cctcctgccc ctcctgctcc t
//
