LOCUS       sequence-173224-       12860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral expression of SpCas9-VQR (NGA PAM restricted).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12860)
  AUTHORS   Canver MC, Lessard S, Pinello L, Wu Y, Ilboudo Y, Stern EN, 
            Needleman AJ, Galacteros F, Brugnara C, Kutlar A, McKenzie C, Reid 
            M, Chen DD, Das PP, A Cole M, Zeng J, Kurita R, Nakamura Y, Yuan GC,
            Lettre G, Bauer DE, Orkin SH
  TITLE     Variant-aware saturating mutagenesis using multiple Cas9 nucleases 
            identifies regulatory elements at trait-associated loci.
  JOURNAL   Nat Genet. 2017 Feb 20. doi: 10.1038/ng.3793.
  PUBMED    28218758
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"; journalName: "Nat Genet. 
            2017 Feb 20. doi: 10.1038/ng.3793."
COMMENT     Sequence Label: lenti-Cas9-VQR-Blast
FEATURES             Location/Qualifiers
     source          1..12860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2651..2862
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2881..2890
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2887..6288,6289..6291,6292..6888,6889..6891,6892..6894,
                     6895..6897,6898..6990)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2887..6990
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6991..7038
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7039..7062
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7072..7128
                     /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             7129..7527
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    7552..8140
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7605..7625)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8023..8034)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8143..8159)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8144..8160)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             8665..8845
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(8871..8888)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8877..9101
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9147..9575
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9234..9253)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9444..9465
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9584..9604)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9589..9918
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9769..9904
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9831..9850
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9966..10013
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10032..10406
                     /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    10536..10657
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10573..10592)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10627..10646
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10706..10722)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10706..10722)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10719..10741)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10730..10746
                     /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(10754..10760,10761..10778,10779..10784))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10799..10820
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10937..10954)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11108..11696)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11188..11207)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11867..12658,12659..12727))
                     /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     12490..12509
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12728..12832)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta agctagctag gtcttgaaag gagtgggaat tggctccggt
     2641 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     2701 ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     2761 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     2821 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagagcgct
     2881 gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     2941 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     3001 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     3061 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     3121 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     3181 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     3241 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     3301 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     3361 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     3421 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     3481 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     3541 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     3601 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     3661 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     3721 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     3781 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     3841 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     3901 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     3961 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     4021 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     4081 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     4141 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     4201 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     4261 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     4321 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     4381 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     4441 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     4501 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     4561 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     4621 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     4681 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     4741 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     4801 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     4861 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     4921 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     4981 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     5041 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     5101 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     5161 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     5221 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     5281 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     5341 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     5401 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     5461 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     5521 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     5581 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     5641 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     5701 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     5761 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     5821 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     5881 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     5941 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     6001 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     6061 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     6121 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     6181 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     6241 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcgt gagccccacc
     6301 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     6361 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     6421 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     6481 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     6541 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     6601 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     6661 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     6721 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     6781 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     6841 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagca gtacagaagc
     6901 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     6961 cggatcgacc tgtctcagct gggaggcgac aagcgacctg ccgccacaaa gaaggctgga
     7021 caggctaaga agaagaaaga ttacaaagac gatgacgata agggatccgg cgcaacaaac
     7081 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgat ggccaagcct
     7141 ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc
     7201 atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact
     7261 ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact
     7321 gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg
     7381 ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc
     7441 aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg
     7501 ccctctggtt atgtgtggga gggctaagaa ttcgatatca agcttatcga taatcaacct
     7561 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     7621 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     7681 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     7741 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     7801 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     7861 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     7921 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     7981 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     8041 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     8101 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga
     8161 gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt
     8221 gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct
     8281 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     8341 ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac
     8401 cacacacaag gctacttccc tgattggcag aactacacac cagggccagg gatcagatat
     8461 ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa ggtagaagaa
     8521 gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg gatggatgac
     8581 ccggagagag aagtattaga gtggaggttt gacagccgcc tagcatttca tcacatggcc
     8641 cgagagctgc atccggactg tactgggtct ctctggttag accagatctg agcctgggag
     8701 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     8761 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     8821 tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag cctcgactgt
     8881 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     8941 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     9001 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     9061 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     9121 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     9181 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     9241 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     9301 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     9361 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     9421 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     9481 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     9541 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     9601 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     9661 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     9721 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     9781 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     9841 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     9901 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag
     9961 cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
    10021 ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
    10081 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
    10141 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
    10201 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
    10261 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
    10321 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
    10381 acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct
    10441 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
    10501 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
    10561 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
    10621 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
    10681 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
    10741 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
    10801 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
    10861 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
    10921 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
    10981 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
    11041 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
    11101 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
    11161 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
    11221 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
    11281 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
    11341 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
    11401 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
    11461 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
    11521 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
    11581 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
    11641 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
    11701 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
    11761 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
    11821 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
    11881 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
    11941 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
    12001 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
    12061 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
    12121 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
    12181 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
    12241 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
    12301 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
    12361 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
    12421 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
    12481 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
    12541 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
    12601 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
    12661 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
    12721 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
    12781 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
    12841 cccgaaaagt gccacctgac
//
