pCMV-PE5max-pGL3-U6--sgRNA-PGK-puroR-(HindIII-XmaI)

LOCUS       pCMV-PE5max-pGL3-U6--sgRNA-PGK-puroR-(HindIII-XmaI)             14542 bp DNA     circular SYN 07-NOV-2025
DEFINITION  expresses sgRNA from the U6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14542)
  AUTHORS   Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins 
            A, Iyer V, Huang X, Skarnes WC
  TITLE     Efficient genome modification by CRISPR-Cas9 nickase with minimal 
            off-target effects.
  JOURNAL   Nat Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857.
  PUBMED    24584192
REFERENCE   2  (bases 1 to 14542)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 19, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857."
FEATURES             Location/Qualifiers
     source          1..14542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(287..312)
                     /label=OL533
     primer_bind     303..348
                     /label=OL551
     primer_bind     complement(341..377)
                     /label=OL552
     misc_RNA        358..433
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(358..372)
                     /label=OL534
     misc_feature    485..602
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        651..1161
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1080..1098
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1183..1782
                     /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(1183..1202)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1679..1699
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1929..2050
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1940..1959)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1994..2013
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2136..2178)
                     /label=PE009
     primer_bind     2159..2206
                     /label=PE007
     enhancer        2163..2542
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2543..2746
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2697..2717
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2788..2807
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2788..2806
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2856..2876
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2877..6977
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from 
                     theStreptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(R221K N394K H840A)
                     /note="generates RNA-guided single strand nicks in DNA
                     dueto the H840A mutation in the HNH catalytic domain"
                     /note="/Original_Translation=DKKYSIGLDIGTNSVGWAVITDEYKVPSKK
                     FKVLG 
                     NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD 
                     DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR 
                     LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA 
                     ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK 
                     DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYD 
                     EHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD 
                     GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
                     ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
                     NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK 
                     VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL 
                     EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQ 
                     SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                     IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE 
                     LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLK 
                     DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG 
                     GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS 
                     DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI 
                     AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF 
                     ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT 
                     VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII 
                     KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ 
                     KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL 
                     FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /note="/modified_by=chenp"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6978..7079
                     /label=SGGSx2-sv40NLS-SGGSx2
                     /note="/created_by=chenp"
     CDS             7080..9110
                     /label=RT codon optimized Genscript
                     /note="/created_by=jessiedavis"
     CDS             9156..9176
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9186..9212
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             9222..9278
                     /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"
     primer_bind     9222..9245
                     /label=PE001
     primer_bind     complement(9260..9279)
                     /label=PE002
     CDS             9279..11534
                     /label=MLH1 del754-756 genscript codon
                     /note="/created_by=chenp"
                     /note="/modified_by=chenp"
     primer_bind     9320..9362
                     /label=OL530
     primer_bind     9333..9368
                     /label=OL532
     misc_feature    9356
                     /label=A to G
     primer_bind     complement(9639..9677)
                     /label=OL531
     CDS             11543..11560
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(11583..11600)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11589..11813
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(11794..11817)
                     /label=PE008
     primer_bind     11801..11841
                     /label=PE010
     primer_bind     complement(11874..11891)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12045..12633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12125..12144)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12804..13664)
                     /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     13427..13446
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13665..13769)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      13796..14251
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13883..13902)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     14093..14114
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    14382..14430
                     /note="synthetic polyadenylation signal"
     misc_feature    14444..14535
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     14484..14503
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacaag cttaccgtga caccggtcag agagggtctc ccgggcagtt
      361 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      421 accgagtcgg tgcttttttt aaagaattct cgacctcgag acaaatggca gtattcatcc
      481 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
      541 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
      601 ttcgggttta ttacagggac agcagagatc cactttggcc gcggctcgag ggggttgggg
      661 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt
      721 tccgggaaac gcagcggcgc cgaccctggg actcgcacat tcttcacgtc cgttcgcagc
      781 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc
      841 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca
      901 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg
      961 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa
     1021 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg
     1081 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga
     1141 atcaccgacc tctctcccca gggggatcca ccggagctta ccatgaccga gtacaagccc
     1201 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     1261 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     1321 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     1381 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     1441 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     1501 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     1561 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     1621 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggaaacctc cgcgccccgc
     1681 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     1741 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg
     1801 cagcgcccga ccgaaaggag cgcacgaccc catgcatcgg tacctttaag accaatgact
     1861 tacaaggcag ctgtagatct tagccacttt ctagagtcgg ggcggccggc cgcttcgagc
     1921 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     1981 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     2041 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     2101 ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatccgtc
     2161 gagacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     2221 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2281 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2341 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2401 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     2461 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     2521 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     2581 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     2641 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     2701 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
     2761 tcagatccgc tagagatccg cggccgctaa tacgactcac tatagggaga gccgccacca
     2821 tgaaacggac agccgacgga agcgagttcg agtcaccaaa gaagaagcgg aaagtcgaca
     2881 agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg
     2941 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
     3001 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc
     3061 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
     3121 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
     3181 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
     3241 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
     3301 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
     3361 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     3421 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     3481 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agaaagctgg
     3541 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg
     3601 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     3661 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     3721 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     3781 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     3841 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     3901 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     3961 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     4021 tggacggcac cgaggaactg ctcgtgaagc tgaagagaga ggacctgctg cggaagcagc
     4081 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     4141 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     4201 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     4261 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     4321 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     4381 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg
     4441 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     4501 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     4561 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     4621 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     4681 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     4741 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     4801 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     4861 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     4921 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     4981 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     5041 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     5101 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     5161 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     5221 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     5281 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     5341 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac gctatcgtgc
     5401 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga
     5461 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     5521 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg
     5581 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     5641 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     5701 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     5761 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     5821 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     5881 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     5941 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     6001 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     6061 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg
     6121 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     6181 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa
     6241 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     6301 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     6361 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     6421 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     6481 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     6541 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     6601 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     6661 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     6721 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca
     6781 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     6841 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     6901 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     6961 ctcagctggg aggtgactcc ggcggaagct ctggtggcag caagcggacc gccgacggct
     7021 ctgaattcga gagccctaag aagaaaagaa aggtgagcgg aggctctagc ggcggaagca
     7081 ccctgaacat tgaagacgag tatagactgc atgaaacaag caaggaaccc gacgtgtccc
     7141 tgggctccac ctggctgtcc gactttcccc aggcctgggc cgagacagga ggaatgggcc
     7201 tggccgtgcg gcaggcaccc ctgatcatcc ctctgaaggc cacctctaca cccgtgagca
     7261 tcaagcagta ccctatgtct caggaggcca gactgggcat caagcctcac atccagaggc
     7321 tgctggacca gggcatcctg gtgccatgcc agagcccctg gaacacacca ctgctgcccg
     7381 tgaagaagcc aggcaccaat gactatagac ccgtgcagga tctgagagag gtgaacaaga
     7441 gggtggagga tatccacccc accgtgccca acccttacaa tctgctgtcc ggcctgcccc
     7501 cttctcacca gtggtataca gtgctggacc tgaaggatgc cttcttttgt ctgagactgc
     7561 accctaccag ccagccactg ttcgcctttg agtggaggga ccctgagatg ggcatctctg
     7621 gccagctgac ctggacacgc ctgcctcagg gcttcaagaa tagcccaaca ctgtttaacg
     7681 aggccctgca ccgcgacctg gcagatttcc ggatccagca cccagatctg atcctgctgc
     7741 agtacgtgga cgatctgctg ctggccgcca ccagcgagct ggattgccag cagggaacac
     7801 gcgccctgct gcagaccctg ggaaacctgg gatatagggc atccgccaag aaggcccaga
     7861 tctgtcagaa gcaggtgaag tacctgggct atctgctgaa ggagggccag agatggctga
     7921 cagaggccag gaaggagaca gtgatgggcc agccaacacc caagacccca agacagctga
     7981 gggagttcct gggcaaagca ggattttgca ggctgttcat cccaggattc gcagagatgg
     8041 cagcacctct gtacccactg accaagccgg gcaccctgtt taattggggc cctgaccagc
     8101 agaaggccta tcaggagatc aagcaggccc tgctgacagc accagccctg ggcctgccag
     8161 acctgaccaa gcctttcgag ctgtttgtgg atgagaagca gggctacgcc aagggcgtgc
     8221 tgacccagaa gctgggacca tggagacggc ccgtggccta tctgtccaag aagctggacc
     8281 cagtggcagc aggatggcca ccatgcctga ggatggtggc agcaatcgcc gtgctgacaa
     8341 aggatgccgg caagctgacc atgggacagc cactggtcat cctggcacca cacgcagtgg
     8401 aggccctggt gaagcagcct ccagatcgct ggctgtctaa cgcccggatg acacactacc
     8461 aggccctgct gctggacacc gatcgcgtgc agtttggccc tgtggtggcc ctgaatccag
     8521 ccaccctgct gcctctgcca gaggagggcc tgcagcacaa ctgtctggac atcctggcag
     8581 aggcacacgg aacaaggcca gacctgaccg atcagcccct gcctgacgcc gatcacacat
     8641 ggtataccga tggaagctcc ctgctgcagg agggccagag gaaggcagga gcagcagtga
     8701 ccacagagac agaagtgatc tgggccaagg ccctgccagc aggcacatcc gcccagcggg
     8761 ccgagctgat cgccctgacc caggccctga agatggccga gggcaagaag ctgaacgtgt
     8821 acacagactc cagatatgcc ttcgccaccg cacacatcca cggagagatc tacaggcgcc
     8881 ggggctggct gacctctgag ggcaaggaga tcaagaacaa ggatgagatc ctggccctgc
     8941 tgaaggccct gtttctgccc aagcggctga gcatcatcca ctgtcctgga caccagaagg
     9001 gacactccgc cgaggcaagg ggcaatcgga tggccgacca ggccgccaga aaggctgcta
     9061 ttactgaaac tcccgacact tccactctgc tgattgaaaa ctcctcccct tctggcggct
     9121 caaaaagaac cgccgacggc agcgaattcg agtctcccaa gaagaagagg aaagtcggct
     9181 ctggccctgc cgctaagaga gtgaagctgg acggaagcgg agctactaac ttcagcctgc
     9241 tgaagcaggc tggagacgtg gaggagaacc ctggacctag cttcgttgct ggagtcatcc
     9301 ggagactgga cgagacagtg gtgaacagaa ttgccgccgg cgaggtgatc cagagacctg
     9361 ccaatgcaat aaaggagatg atcgagaact gtctggacgc caagtccaca agcattcagg
     9421 tgatcgtgaa ggagggcgga ctgaagctga tccagatcca agacaacggc acaggcatca
     9481 gaaaggaaga tctggacatc gtgtgtgaac ggttcaccac atctaagctg cagtcttttg
     9541 aggatctggc ctctatcagt acctacggct tcagaggcga ggccctggcc agcatcagcc
     9601 acgtggccca tgtgaccatc accaccaaaa ccgccgacgg caaatgcgct tatcgcgcta
     9661 gctacagcga cggcaagctg aaagccccgc caaagccttg cgccggcaac cagggtacac
     9721 agataacagt ggaggatctg ttctacaaca tcgccacccg gagaaaggcc ctgaaaaatc
     9781 ccagcgagga gtacggcaag atcctggaag tcgtcggcag atactccgtg cacaacgccg
     9841 gaatcagctt tagcgtaaag aagcagggag aaaccgtggc cgatgtgcgc accctgccaa
     9901 atgccagcac cgtggataac atcagaagca ttttcggaaa tgccgtgtcc agagaactga
     9961 tcgagatcgg ctgcgaagat aagaccctgg cttttaagat gaacggctac atcagcaacg
    10021 ccaattactc tgtgaagaag tgcatctttc ttctgttcat caaccacaga ctggtggaaa
    10081 gcaccagcct gcggaaagcc atcgagacag tgtacgccgc ctacctgcct aagaacaccc
    10141 accccttcct gtacctgagc ctcgagatca gccctcagaa cgtggacgtc aatgtgcatc
    10201 ctacaaagca cgaggtgcac ttcctgcacg aggaaagcat cctggaaaga gtgcagcagc
    10261 acattgagag caagctgctg ggctctaaca gcagcagaat gtacttcaca cagaccctgc
    10321 tgcctggcct ggccggcccc tcaggcgaaa tggttaagtc cacaacctct ctgacctcat
    10381 ccagcaccag cggttcttcc gataaggtgt acgcccacca gatggtgcgg accgactctc
    10441 gggagcagaa gctggacgcc tttctgcaac ctctgagcaa acctctgagc tctcagcctc
    10501 aggccatcgt gaccgaggac aagacagata tctcctccgg ccgtgccaga cagcaggacg
    10561 aagaaatgct cgagctgcca gctcctgccg aggtggccgc caagaaccag agcctggagg
    10621 gagataccac aaagggcacc agcgaaatga gcgagaagcg gggccctacc tccagcaacc
    10681 ccagaaaacg gcaccgggag gacagcgacg tggaaatggt ggaggacgac agcagaaagg
    10741 aaatgacagc cgcttgtacc cctagaagaa gaatcatcaa cctgacctcc gtgctgagcc
    10801 tgcaggagga gatcaacgag cagggccacg aggtgctgag agagatgctg cacaatcaca
    10861 gcttcgtggg ctgcgtgaac cctcaatggg ccctggctca gcatcaaaca aagctgtacc
    10921 tgctgaacac caccaagctg agcgaagagc tgttctacca gatcctcatc tacgacttcg
    10981 ccaacttcgg cgtgctacgc ctgagcgagc ccgcccctct gtttgacctg gccatgctgg
    11041 ctctggatag cccagaaagc ggctggacag aagaggacgg acctaaagag gggctggctg
    11101 aatacatcgt ggagttcctg aagaaaaagg ccgagatgct ggccgactac ttttctctgg
    11161 aaatcgacga ggaaggcaac ctgatcggcc tgcctctgct gatcgataac tacgtgcctc
    11221 ccctggaagg cctgcccatc ttcatcctga gactggctac agaggtgaac tgggacgagg
    11281 aaaaggaatg cttcgagtct ctgagcaagg agtgcgccat gttctatagc atcagaaaac
    11341 agtacatctc tgaagagagc actctgtctg gccagcagag tgaagtgccc ggaagcatcc
    11401 ccaacagctg gaagtggacc gtggaacaca tcgtgtacaa ggccctgcgg agccacattc
    11461 tccctcctaa gcacttcacc gaggacggca acatcctgca gctggccaac ctgcccgacc
    11521 tttataaggt tttctaaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc
    11581 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
    11641 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgaga aaattgcatc
    11701 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
    11761 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggatgactg
    11821 tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc ttcctcgctc
    11881 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
    11941 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
    12001 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
    12061 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
    12121 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
    12181 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
    12241 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
    12301 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
    12361 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
    12421 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
    12481 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
    12541 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
    12601 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
    12661 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
    12721 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
    12781 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
    12841 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
    12901 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgggaccc acgctcaccg
    12961 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
    13021 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
    13081 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
    13141 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
    13201 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
    13261 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
    13321 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
    13381 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
    13441 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
    13501 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
    13561 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
    13621 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
    13681 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
    13741 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg
    13801 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
    13861 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
    13921 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
    13981 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
    14041 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
    14101 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
    14161 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
    14221 aattttaaca aaatattaac gcttacaatt tgccattcgc cattcaggct gcgcaactgt
    14281 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agcccaagct accatgataa
    14341 gtaagtaata ttaaggtacg ggaggtactt ggagcggccg caataaaata tctttatttt
    14401 cattacatct gtgtgttggt tttttgtgtg aatcgatagt actaacatac gctctccatc
    14461 aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca agtgcaggtg
    14521 ccagaacatt tctctatcga ta
//