LOCUS       sequence-181681-       12860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized S. pyogenes Cas9n (D10A nickase) 
            protein and blasticidin resistance from EFS promoter. Lentiviral 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12860)
  AUTHORS   Sanjana NE, Shalem O, Zhang F
  TITLE     Improved vectors and genome-wide libraries for CRISPR screening.
  JOURNAL   Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.
  PUBMED    25075903
REFERENCE   2  (bases 1 to 12860)
  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: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2014-08"; volume: "11"; issue: 
            "8"; pages: "783-4"
COMMENT     Sequence Label: lentiCas9n(D10A)-Blast
FEATURES             Location/Qualifiers
     source          1..12860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(3..23)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8..337
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      188..323
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     250..269
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        385..432
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             451..825
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    955..1076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(992..1011)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1046..1065
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1125..1141)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1125..1141)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1138..1160)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1149..1165
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1173..1179,1180..1197,1198..1203))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1218..1239
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1356..1373)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1527..2115)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1607..1626)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2286..3077,3078..3146))
                     /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     2909..2928
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3147..3251)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3326..3345)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3517..3896
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3898..4096
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4051..4071
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4114..4294
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4341..4466
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4959..5192
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5377..5421
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    5719..5836
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5930..6141
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6160..6169
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6166..6192,6193..6195,6196..10269)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6166..10269
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             10270..10317
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10318..10341
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             10351..10407
                     /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"
     CDS             10408..10806
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    10831..11419
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10884..10904)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11302..11313)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11422..11438)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11423..11439)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11944..12124
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(12150..12167)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12156..12380
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12426..12854
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12513..12532)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12723..12744
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
       61 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
      121 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
      181 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
      241 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
      301 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
      361 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
      421 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
      481 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
      541 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
      601 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
      661 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
      721 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
      781 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat
      841 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
      901 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
      961 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     1021 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     1081 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     1141 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     1201 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     1261 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1321 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     1381 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     1441 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1501 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1561 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1621 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1681 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1741 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1801 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1861 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1921 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     1981 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2041 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2101 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2161 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     2221 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2281 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     2341 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     2401 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     2461 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     2521 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     2581 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     2641 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     2701 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     2761 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     2821 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     2881 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     2941 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3001 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3061 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3121 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     3181 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3241 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     3301 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3361 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     3421 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     3481 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
     3541 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     3601 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     3661 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     3721 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     3781 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     3841 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     3901 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     3961 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     4021 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     4081 aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga
     4141 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     4201 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     4261 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
     4321 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
     4381 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
     4441 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
     4501 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
     4561 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
     4621 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     4681 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     4741 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     4801 aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa
     4861 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag
     4921 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc
     4981 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga
     5041 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
     5101 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct
     5161 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc
     5221 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt
     5281 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata
     5341 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa
     5401 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
     5461 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
     5521 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
     5581 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga
     5641 gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat
     5701 ggctgtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
     5761 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     5821 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt ggttaattaa
     5881 gctagctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc
     5941 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag
     6001 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
     6061 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
     6121 gggtttgccg ccagaacaca ggaccggttc tagagcgctg ccaccatgga caagaagtac
     6181 agcatcggcc tggccatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     6241 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     6301 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     6361 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     6421 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     6481 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     6541 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     6601 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     6661 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     6721 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     6781 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     6841 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gcaacctgat tgccctgagc
     6901 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     6961 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     7021 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     7081 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     7141 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     7201 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     7261 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     7321 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     7381 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     7441 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     7501 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     7561 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     7621 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     7681 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     7741 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     7801 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     7861 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     7921 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     7981 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     8041 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     8101 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     8161 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     8221 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     8281 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     8341 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     8401 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     8461 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     8521 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     8581 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     8641 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     8701 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     8761 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     8821 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     8881 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     8941 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     9001 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     9061 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     9121 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     9181 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     9241 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     9301 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     9361 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     9421 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     9481 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     9541 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     9601 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     9661 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     9721 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     9781 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     9841 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     9901 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     9961 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
    10021 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
    10081 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
    10141 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
    10201 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
    10261 ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa gaagaaagat
    10321 tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct gaaacaagcc
    10381 ggagatgtcg aagagaatcc tggaccgatg gccaagcctt tgtctcaaga agaatccacc
    10441 ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc
    10501 gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt
    10561 actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc
    10621 aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga
    10681 cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt
    10741 gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag
    10801 ggctaagaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
    10861 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
    10921 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
    10981 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
    11041 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
    11101 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
    11161 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
    11221 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
    11281 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
    11341 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
    11401 ctttgggccg cctccccgca tcgataccgt cgacctcgag acctagaaaa acatggagca
    11461 atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag
    11521 gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag
    11581 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
    11641 tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct
    11701 gattggcaga actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc
    11761 tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg agagaacacc
    11821 cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag
    11881 tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca tccggactgt
    11941 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
    12001 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
    12061 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
    12121 agcagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
    12181 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
    12241 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
    12301 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
    12361 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
    12421 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
    12481 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
    12541 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
    12601 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
    12661 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
    12721 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
    12781 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
    12841 atttaacgcg aattaattct
//
