LOCUS       sequence-423158-       14684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Cas9 and sgRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14684)
  AUTHORS   Habashy et al
  TITLE     CRISPR/Cas9-based system for loss-of-function screening
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 14684)
  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: 
            "Unpublished"
COMMENT     Sequence Label: lentiCRISPR v2-Blast
FEATURES             Location/Qualifiers
     source          1..14684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             857..904
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             905..928
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             938..994
                     /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             995..1393
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    1418..2006
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1471..1491)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1889..1900)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2078..2311
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(2337..2354)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2343..2567
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2613..3041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2700..2719)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2910..2931
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3050..3070)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3055..3384
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3235..3370
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3297..3316
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3432..3479
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3498..3872
                     /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    4002..4123
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4039..4058)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4093..4112
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4172..4188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4172..4188)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4185..4207)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4196..4212
                     /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(4220..4226,4227..4244,4245..4250))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4265..4286
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4403..4420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4574..5162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4654..4673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5333..6124,6125..6193))
                     /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     5956..5975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6194..6298)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6373..6392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6564..6943
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6945..7143
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7098..7118
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             7161..7341
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7388..7513
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8006..8239
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8424..8468
                     /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    8766..8883
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8934..9182
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8934..8954
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     9105..9124
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        11064..11139
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        11201..11412
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      11431..11440
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(11437..14684,1..856)
                     /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
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(11437..14684,1..856)
                     /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
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
       61 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
      121 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
      181 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
      241 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
      301 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
      361 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
      421 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
      481 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
      541 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
      601 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
      661 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
      721 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
      781 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
      841 tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa
      901 gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa
      961 acaagccgga gatgtcgaag agaatcctgg accgatggcc aagcctttgt ctcaagaaga
     1021 atccaccctc attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta
     1081 cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata
     1141 tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc
     1201 agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc
     1261 ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa
     1321 ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt
     1381 gtgggagggc taagaattcg atatcaagct tatcggtaat caacctctgg attacaaaat
     1441 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     1501 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     1561 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     1621 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     1681 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     1741 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     1801 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     1861 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     1921 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     1981 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     2041 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     2101 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     2161 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     2221 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     2281 cccttttagt cagtgtggaa aatctctagc agggcccgtt taaacccgct gatcagcctc
     2341 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2401 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2461 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2521 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2581 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2641 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2701 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2761 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2821 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2881 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2941 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3001 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3061 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3121 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3181 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3241 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3301 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3361 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     3421 gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca
     3481 aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg
     3541 acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg
     3601 aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg
     3661 accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt
     3721 acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga
     3781 ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact
     3841 gcgtgcactt cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg
     3901 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     3961 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4021 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4081 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4141 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4201 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4261 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4321 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4381 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4441 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4501 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4561 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4621 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4681 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4741 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4801 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4861 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4921 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4981 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5041 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5101 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5161 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5221 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5281 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5341 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5401 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5461 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5521 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5581 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5641 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5701 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5761 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5821 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5881 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5941 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6001 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6061 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6121 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6181 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6241 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6301 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     6361 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     6421 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     6481 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     6541 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     6601 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     6661 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     6721 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     6781 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     6841 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     6901 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     6961 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     7021 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     7081 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     7141 cagcgcgttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     7201 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     7261 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     7321 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
     7381 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
     7441 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
     7501 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
     7561 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     7621 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     7681 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     7741 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     7801 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga
     7861 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     7921 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     7981 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     8041 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     8101 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     8161 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     8221 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     8281 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     8341 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     8401 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     8461 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     8521 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata
     8581 gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
     8641 ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc
     8701 gattagtgaa cggatcggca ctgcgtgcgc caattctgca gacaaatggc agtattcatc
     8761 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     8821 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     8881 tttcgggttt attacaggga cagcagagat ccagtttggt taattaaggt accgagggcc
     8941 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta
     9001 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     9061 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     9121 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     9181 ccggagacgg ttgtaaatga gcacacaaaa tacacatgct aaaatattat attctatgac
     9241 ctttataaaa tcaaccaaaa tcttcttttt aataacttta gtatcaataa ttagaatttt
     9301 tatgttcctt tttgcaaact tttaataaaa atgagcaaaa taaaaaaacg ctagttttag
     9361 taactcgcgt tgttttcttc acctttaata atagctactc caccacttgt tcctaagcgg
     9421 tcagctcctg cttcaatcat tttttgagca tcttcaaatg ttctaactcc accagctgct
     9481 ttaactaaag cattgtcttt aacaactgac ttcattagtt taacatcttc aaatgttgca
     9541 cctgattttg aaaatcctgt tgatgtttta acaaattcta atccagcttc aacagctatt
     9601 tcacaagctt tcatgatttc ttcttttgtt aataaacaat tttccataat acatttaaca
     9661 acatgtgatc cagctgcttt ttttacagct ttcatgtctt ctaaaactaa ttcataattt
     9721 ttgtctttta atgcaccaat atttaatacc atatcaattt ctgttgcacc atctttaatt
     9781 gcttcagaaa cttcgaatgc ttttgtagct gttgtgcatg cacctagagg aaaacctaca
     9841 acatttgtta ttcctacatt tgtgcctttt aataattctt tacaatagct tgttcaatat
     9901 gaattaacac aaactgttgc aaaatcaaat tcaattgctt catcacataa ttgtttaatt
     9961 tcagctttcg tagcatcttg ttttaataat gtgtgatcta tatatttgtt tagtttcatt
    10021 ttttctccta tatattcatt tttaatttta attctttaat aatttcgtct actttaactt
    10081 tagcgttttg aacagattca ccaacaccta taaaataaat ttttagttta ggttcagttc
    10141 cacttgggcg aacagcaaat catgacttat cttctaaata aaattttagt aagtcttgtc
    10201 ctggcatatt atacattcca tcgatgtagt cttcaacatt aacaacttta agtccagcaa
    10261 tttgagttaa gggtgttgct ctcaatgatt tcattaatgg ttcaattttt aatttctttt
    10321 cttctggttt aaaattcaag tttaaagtga aagtgtaata tgcacccatt tctttaaata
    10381 aatcttctaa atagtctact aatgttttat tttgtttttt ataaaatcaa gcagcctctg
    10441 ctattaatat agaagcttgt attccatctt tatctctagc tgagtcatca attacatatc
    10501 cataactttc ttcataagca aaaacaaaat ttaatccgtt atcttcttct ttagcaattt
    10561 ctctacccat tcatttaaat ccagttaaag tttttacaat attaactcca tatttttcat
    10621 gagcgattct atcacccaaa tcacttgtta caaaacttga atatagagcc ggattttttg
    10681 gaatgctatt taagcgtttt agatttgata attttcaatc aattaaaatt ggtcctgttt
    10741 gatttccatc taatcttaca aaatgaccat catgttttat tgccattcca aatctgtcag
    10801 catctgggtc attcataata ataatatctg catcatgttt aataccatat tcaagcggta
    10861 tttttcatgc aggatcaaat tctggatttg gatttacaac atttttaaat gtttcatctt
    10921 caaatgcatg ctcttcaacc tcaataacgt tatatcctga ttcacgtaat atttttgggg
    10981 taaatttagt tcctgttcca ttaactgcgc taaaaataat ttttaaatct tttttagctt
    11041 cttgctcttt tttgtacgtc tctgttttag agctagaaat agcaagttaa aataaggcta
    11101 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgaatt cgctagctag
    11161 gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc
    11221 acagtccccg agaagttggg gggaggggtc ggcaattgat ccggtgccta gagaaggtgg
    11281 cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg
    11341 ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc
    11401 gccagaacac aggaccggtt ctagagcgct gccaccatgg acaagaagta cagcatcggc
    11461 ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
    11521 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
    11581 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
    11641 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
    11701 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
    11761 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
    11821 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
    11881 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
    11941 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
    12001 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
    12061 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
    12121 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg
    12181 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
    12241 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
    12301 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
    12361 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
    12421 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
    12481 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
    12541 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
    12601 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
    12661 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
    12721 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
    12781 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
    12841 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
    12901 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
    12961 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
    13021 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
    13081 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
    13141 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
    13201 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
    13261 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
    13321 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
    13381 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac
    13441 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
    13501 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
    13561 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
    13621 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
    13681 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
    13741 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
    13801 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
    13861 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
    13921 gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag
    13981 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac
    14041 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac
    14101 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg
    14161 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
    14221 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag
    14281 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
    14341 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
    14401 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
    14461 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
    14521 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
    14581 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
    14641 accggggaga tcgtgtggga taagggccgg gattttgcca ccgt
//
