pCMV-PE5max-pGL3-U6-(HindIII-XmaI)-Scaffold-PGK-puroR-EGFP

LOCUS       pCMV-PE5max-pGL3-U6-(HindIII-XmaI)-Scaffold-PGK-puroR-EGFP              15316 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 15316)
  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 15316)
  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..15316
                     /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     935..951
                     /label=OL446
     primer_bind     1080..1098
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1183..1779
                     /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"
     primer_bind     complement(1765..1787)
                     /label=PE006
     primer_bind     1772..1803
                     /label=PE001
     CDS             1780..1836
                     /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     complement(1818..1844)
                     /label=PE002
     primer_bind     1828..1854
                     /label=PE003
     CDS             1837..2556
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2533..2563)
                     /label=PE004
     primer_bind     2549..2570
                     /label=PE005
     polyA_signal    2703..2824
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2714..2733)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2768..2787
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2910..2952)
                     /label=PE009
     primer_bind     2933..2980
                     /label=PE007
     enhancer        2937..3316
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3317..3520
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3471..3491
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3562..3581
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3562..3580
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3630..3650
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3651..7751
                     /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             7752..7853
                     /label=SGGSx2-sv40NLS-SGGSx2
                     /note="/created_by=chenp"
     CDS             7854..9884
                     /label=RT codon optimized Genscript
                     /note="/created_by=jessiedavis"
     primer_bind     9745..9766
                     /label=test
     primer_bind     9818..9840
                     /label=OL447
     CDS             9930..9950
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9960..9986
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             9996..10052
                     /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     9996..10019
                     /label=PE001
     primer_bind     complement(10034..10053)
                     /label=PE002
     CDS             10053..12308
                     /label=MLH1 del754-756 genscript codon
                     /note="/created_by=chenp"
                     /note="/modified_by=chenp"
     primer_bind     10094..10136
                     /label=OL530
     primer_bind     10107..10142
                     /label=OL532
     primer_bind     10115..10145
                     /label=OL485
     primer_bind     complement(10115..10145)
                     /label=OL486
     misc_feature    10130
                     /label=A to G
     primer_bind     complement(10413..10451)
                     /label=OL531
     CDS             12317..12334
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(12357..12374)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12363..12587
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(12568..12591)
                     /label=PE008
     primer_bind     12575..12615
                     /label=PE010
     primer_bind     complement(12648..12665)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12819..13407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12899..12918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(13578..14438)
                     /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     14201..14220
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14439..14543)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      14570..15025
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(14657..14676)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     14867..14888
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    15156..15204
                     /note="synthetic polyadenylation signal"
     misc_feature    15218..15309
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     15258..15277
                     /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 cccggtgccg ctactaactt cagcctgctg
     1801 aagcaggctg gagacgtgga ggagaaccct ggacctatgg tgagcaaggg cgaggagctg
     1861 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1921 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     1981 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     2041 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2101 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2161 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2221 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     2281 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     2341 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     2401 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     2461 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2521 gggatcactc tcggcatgga cgagctgtac aagtaacgcc cgccccacga cccgcagcgc
     2581 ccgaccgaaa ggagcgcacg accccatgca tcggtacctt taagaccaat gacttacaag
     2641 gcagctgtag atcttagcca ctttctagag tcggggcggc cggccgcttc gagcagacat
     2701 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     2761 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     2821 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     2881 tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtcgagaca
     2941 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     3001 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     3061 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     3121 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     3181 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     3241 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     3301 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     3361 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     3421 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     3481 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
     3541 ccgctagaga tccgcggccg ctaatacgac tcactatagg gagagccgcc accatgaaac
     3601 ggacagccga cggaagcgag ttcgagtcac caaagaagaa gcggaaagtc gacaagaagt
     3661 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
     3721 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
     3781 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
     3841 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
     3901 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
     3961 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
     4021 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
     4081 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
     4141 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
     4201 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
     4261 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagaaag ctggaaaatc
     4321 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga
     4381 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
     4441 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
     4501 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
     4561 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
     4621 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
     4681 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
     4741 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
     4801 gcaccgagga actgctcgtg aagctgaaga gagaggacct gctgcggaag cagcggacct
     4861 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
     4921 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
     4981 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
     5041 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
     5101 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
     5161 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
     5221 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
     5281 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
     5341 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
     5401 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
     5461 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
     5521 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
     5581 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
     5641 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
     5701 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
     5761 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
     5821 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     5881 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     5941 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     6001 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     6061 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     6121 accaggaact ggacatcaac cggctgtccg actacgatgt ggacgctatc gtgcctcaga
     6181 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     6241 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     6301 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     6361 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     6421 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     6481 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     6541 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     6601 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     6661 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     6721 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     6781 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     6841 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     6901 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     6961 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     7021 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     7081 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     7141 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     7201 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     7261 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     7321 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     7381 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     7441 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     7501 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     7561 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     7621 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     7681 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     7741 tgggaggtga ctccggcgga agctctggtg gcagcaagcg gaccgccgac ggctctgaat
     7801 tcgagagccc taagaagaaa agaaaggtga gcggaggctc tagcggcgga agcaccctga
     7861 acattgaaga cgagtataga ctgcatgaaa caagcaagga acccgacgtg tccctgggct
     7921 ccacctggct gtccgacttt ccccaggcct gggccgagac aggaggaatg ggcctggccg
     7981 tgcggcaggc acccctgatc atccctctga aggccacctc tacacccgtg agcatcaagc
     8041 agtaccctat gtctcaggag gccagactgg gcatcaagcc tcacatccag aggctgctgg
     8101 accagggcat cctggtgcca tgccagagcc cctggaacac accactgctg cccgtgaaga
     8161 agccaggcac caatgactat agacccgtgc aggatctgag agaggtgaac aagagggtgg
     8221 aggatatcca ccccaccgtg cccaaccctt acaatctgct gtccggcctg cccccttctc
     8281 accagtggta tacagtgctg gacctgaagg atgccttctt ttgtctgaga ctgcacccta
     8341 ccagccagcc actgttcgcc tttgagtgga gggaccctga gatgggcatc tctggccagc
     8401 tgacctggac acgcctgcct cagggcttca agaatagccc aacactgttt aacgaggccc
     8461 tgcaccgcga cctggcagat ttccggatcc agcacccaga tctgatcctg ctgcagtacg
     8521 tggacgatct gctgctggcc gccaccagcg agctggattg ccagcaggga acacgcgccc
     8581 tgctgcagac cctgggaaac ctgggatata gggcatccgc caagaaggcc cagatctgtc
     8641 agaagcaggt gaagtacctg ggctatctgc tgaaggaggg ccagagatgg ctgacagagg
     8701 ccaggaagga gacagtgatg ggccagccaa cacccaagac cccaagacag ctgagggagt
     8761 tcctgggcaa agcaggattt tgcaggctgt tcatcccagg attcgcagag atggcagcac
     8821 ctctgtaccc actgaccaag ccgggcaccc tgtttaattg gggccctgac cagcagaagg
     8881 cctatcagga gatcaagcag gccctgctga cagcaccagc cctgggcctg ccagacctga
     8941 ccaagccttt cgagctgttt gtggatgaga agcagggcta cgccaagggc gtgctgaccc
     9001 agaagctggg accatggaga cggcccgtgg cctatctgtc caagaagctg gacccagtgg
     9061 cagcaggatg gccaccatgc ctgaggatgg tggcagcaat cgccgtgctg acaaaggatg
     9121 ccggcaagct gaccatggga cagccactgg tcatcctggc accacacgca gtggaggccc
     9181 tggtgaagca gcctccagat cgctggctgt ctaacgcccg gatgacacac taccaggccc
     9241 tgctgctgga caccgatcgc gtgcagtttg gccctgtggt ggccctgaat ccagccaccc
     9301 tgctgcctct gccagaggag ggcctgcagc acaactgtct ggacatcctg gcagaggcac
     9361 acggaacaag gccagacctg accgatcagc ccctgcctga cgccgatcac acatggtata
     9421 ccgatggaag ctccctgctg caggagggcc agaggaaggc aggagcagca gtgaccacag
     9481 agacagaagt gatctgggcc aaggccctgc cagcaggcac atccgcccag cgggccgagc
     9541 tgatcgccct gacccaggcc ctgaagatgg ccgagggcaa gaagctgaac gtgtacacag
     9601 actccagata tgccttcgcc accgcacaca tccacggaga gatctacagg cgccggggct
     9661 ggctgacctc tgagggcaag gagatcaaga acaaggatga gatcctggcc ctgctgaagg
     9721 ccctgtttct gcccaagcgg ctgagcatca tccactgtcc tggacaccag aagggacact
     9781 ccgccgaggc aaggggcaat cggatggccg accaggccgc cagaaaggct gctattactg
     9841 aaactcccga cacttccact ctgctgattg aaaactcctc cccttctggc ggctcaaaaa
     9901 gaaccgccga cggcagcgaa ttcgagtctc ccaagaagaa gaggaaagtc ggctctggcc
     9961 ctgccgctaa gagagtgaag ctggacggaa gcggagctac taacttcagc ctgctgaagc
    10021 aggctggaga cgtggaggag aaccctggac ctagcttcgt tgctggagtc atccggagac
    10081 tggacgagac agtggtgaac agaattgccg ccggcgaggt gatccagaga cctgccaatg
    10141 caataaagga gatgatcgag aactgtctgg acgccaagtc cacaagcatt caggtgatcg
    10201 tgaaggaggg cggactgaag ctgatccaga tccaagacaa cggcacaggc atcagaaagg
    10261 aagatctgga catcgtgtgt gaacggttca ccacatctaa gctgcagtct tttgaggatc
    10321 tggcctctat cagtacctac ggcttcagag gcgaggccct ggccagcatc agccacgtgg
    10381 cccatgtgac catcaccacc aaaaccgccg acggcaaatg cgcttatcgc gctagctaca
    10441 gcgacggcaa gctgaaagcc ccgccaaagc cttgcgccgg caaccagggt acacagataa
    10501 cagtggagga tctgttctac aacatcgcca cccggagaaa ggccctgaaa aatcccagcg
    10561 aggagtacgg caagatcctg gaagtcgtcg gcagatactc cgtgcacaac gccggaatca
    10621 gctttagcgt aaagaagcag ggagaaaccg tggccgatgt gcgcaccctg ccaaatgcca
    10681 gcaccgtgga taacatcaga agcattttcg gaaatgccgt gtccagagaa ctgatcgaga
    10741 tcggctgcga agataagacc ctggctttta agatgaacgg ctacatcagc aacgccaatt
    10801 actctgtgaa gaagtgcatc tttcttctgt tcatcaacca cagactggtg gaaagcacca
    10861 gcctgcggaa agccatcgag acagtgtacg ccgcctacct gcctaagaac acccacccct
    10921 tcctgtacct gagcctcgag atcagccctc agaacgtgga cgtcaatgtg catcctacaa
    10981 agcacgaggt gcacttcctg cacgaggaaa gcatcctgga aagagtgcag cagcacattg
    11041 agagcaagct gctgggctct aacagcagca gaatgtactt cacacagacc ctgctgcctg
    11101 gcctggccgg cccctcaggc gaaatggtta agtccacaac ctctctgacc tcatccagca
    11161 ccagcggttc ttccgataag gtgtacgccc accagatggt gcggaccgac tctcgggagc
    11221 agaagctgga cgcctttctg caacctctga gcaaacctct gagctctcag cctcaggcca
    11281 tcgtgaccga ggacaagaca gatatctcct ccggccgtgc cagacagcag gacgaagaaa
    11341 tgctcgagct gccagctcct gccgaggtgg ccgccaagaa ccagagcctg gagggagata
    11401 ccacaaaggg caccagcgaa atgagcgaga agcggggccc tacctccagc aaccccagaa
    11461 aacggcaccg ggaggacagc gacgtggaaa tggtggagga cgacagcaga aaggaaatga
    11521 cagccgcttg tacccctaga agaagaatca tcaacctgac ctccgtgctg agcctgcagg
    11581 aggagatcaa cgagcagggc cacgaggtgc tgagagagat gctgcacaat cacagcttcg
    11641 tgggctgcgt gaaccctcaa tgggccctgg ctcagcatca aacaaagctg tacctgctga
    11701 acaccaccaa gctgagcgaa gagctgttct accagatcct catctacgac ttcgccaact
    11761 tcggcgtgct acgcctgagc gagcccgccc ctctgtttga cctggccatg ctggctctgg
    11821 atagcccaga aagcggctgg acagaagagg acggacctaa agaggggctg gctgaataca
    11881 tcgtggagtt cctgaagaaa aaggccgaga tgctggccga ctacttttct ctggaaatcg
    11941 acgaggaagg caacctgatc ggcctgcctc tgctgatcga taactacgtg cctcccctgg
    12001 aaggcctgcc catcttcatc ctgagactgg ctacagaggt gaactgggac gaggaaaagg
    12061 aatgcttcga gtctctgagc aaggagtgcg ccatgttcta tagcatcaga aaacagtaca
    12121 tctctgaaga gagcactctg tctggccagc agagtgaagt gcccggaagc atccccaaca
    12181 gctggaagtg gaccgtggaa cacatcgtgt acaaggccct gcggagccac attctccctc
    12241 ctaagcactt caccgaggac ggcaacatcc tgcagctggc caacctgccc gacctttata
    12301 aggttttcta accggtcatc atcaccatca ccattgagtt taaacccgct gatcagcctc
    12361 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
    12421 cctggaaggt gccactccca ctgtcctttc ctaataaaat gagaaaattg catcgcattg
    12481 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
    12541 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggatg actgtcttct
    12601 ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac
    12661 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
    12721 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
    12781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
    12841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
    12901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
    12961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
    13021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
    13081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
    13141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
    13201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
    13261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
    13321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
    13381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
    13441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
    13501 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
    13561 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
    13621 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
    13681 ggcttaccat ctggccccag tgctgcaatg ataccgcggg acccacgctc accggctcca
    13741 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
    13801 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
    13861 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
    13921 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
    13981 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
    14041 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
    14101 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
    14161 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
    14221 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
    14281 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
    14341 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
    14401 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
    14461 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
    14521 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt
    14581 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
    14641 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
    14701 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
    14761 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
    14821 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
    14881 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
    14941 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
    15001 aacaaaatat taacgcttac aatttgccat tcgccattca ggctgcgcaa ctgttgggaa
    15061 gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg ataagtaagt
    15121 aatattaagg tacgggaggt acttggagcg gccgcaataa aatatcttta ttttcattac
    15181 atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc catcaaaaca
    15241 aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa
    15301 catttctcta tcgata
//