LOCUS       sequence-405885-       13651 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Lentiviral vector modified to express two sgRNAs that delete the 
            intronic SVA within the SCN8A gene with EF-1apha for Cas9 and BFP 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13651)
  AUTHORS   Nadler MJS, Chang W, Ozkaynak E, Huo Y, Nong Y, Boillot M, Johnson 
            M, Moreno A, Matthew P Anderson
  TITLE     Hominoid SVA-lncRNA AK057321 targets human-specific SVA 
            retrotransposons in SCN8A and CDK5RAP2 to initiate neuronal 
            maturation.
  JOURNAL   Commun Biol. 2023 Mar 30;6(1):347. doi: 10.1038/s42003-023-04683-8.
  PUBMED    36997626
REFERENCE   2  (bases 1 to 13651)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Commun 
            Biol."; date: "2023-03-30"; volume: "6(1):347. doi"; pages: " 
            10.1038/s42003-023-04683-8"
COMMENT     Sequence Label: lentiCRISPRv2-sgRNAs-del-SCN8A-SVA-BFP
FEATURES             Location/Qualifiers
     source          1..13651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      196..624
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(283..302)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     493..514
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(633..653)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        638..967
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      818..953
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     880..899
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        1015..1062
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1081..1455
                     /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    1585..1706
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1622..1641)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1676..1695
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1755..1771)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1755..1771)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1768..1790)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1779..1795
                     /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(1803..1809,1810..1827,1828..1833))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1848..1869
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1986..2003)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2157..2745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2237..2256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2916..3707,3708..3776))
                     /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     3539..3558
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3777..3881)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3956..3975)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4151..4530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4532..4730
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4685..4705
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4748..4928
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4975..5100
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5593..5826
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6011..6055
                     /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    6353..6470
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6573..6821
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        6843..6918
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        7002..7250
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        7272..7347
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        7430..7641
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7660..7669
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7666..11769
                     /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             7666..11769
                     /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             11770..11817
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             11818..11841
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             11851..11907
                     /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             11938..12636
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     misc_feature    12652..13240
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12705..12725)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(13123..13134)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             13312..13545
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(13571..13588)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(13577..13651,1..150)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
       61 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
      121 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
      181 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      241 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      301 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      361 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      421 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      481 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
      541 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
      601 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
      661 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
      721 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
      781 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
      841 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
      901 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
      961 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac
     1021 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
     1081 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
     1141 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
     1201 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
     1261 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
     1321 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
     1381 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
     1441 gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg
     1501 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     1561 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     1621 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     1681 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     1741 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1801 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1861 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1921 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1981 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2041 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2101 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2161 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2221 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2281 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2341 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2401 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2461 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2521 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2581 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2641 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2701 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2761 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2821 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2881 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2941 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3001 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3061 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3121 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3181 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3241 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3301 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3361 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3421 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3481 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3541 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3601 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3661 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3721 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3781 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3841 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3901 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     3961 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     4021 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     4081 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     4141 cgcgcgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     4201 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     4261 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     4321 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     4381 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     4441 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     4501 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     4561 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     4621 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     4681 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc
     4741 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     4801 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     4861 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     4921 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
     4981 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
     5041 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
     5101 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
     5161 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
     5221 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     5281 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     5341 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     5401 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg
     5461 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
     5521 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
     5581 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
     5641 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
     5701 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
     5761 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
     5821 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
     5881 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
     5941 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
     6001 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
     6061 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
     6121 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg
     6181 atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga
     6241 aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg
     6301 atcggcactg cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa
     6361 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     6421 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     6481 acagggacag cagagatcca gtttggttaa ttaaggtacc ttcgggttta ttacagggac
     6541 agcagagatc cagtttggtt aattaaggta ccgagggcct atttcccatg attccttcat
     6601 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
     6661 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     6721 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     6781 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggggatctt agaagggatg
     6841 cagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     6901 gtggcaccga gtcggtgctt ttttgaattc gctagctagg tcttgaaagg agtgggaatt
     6961 ggctccggtg cccgtcagtg ggcagagcgc acatcctcga ggagggccta tttcccatga
     7021 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga
     7081 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     7141 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     7201 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gagtctacag
     7261 gatgccacta agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     7321 cttgaaaaag tggcaccgag tcggtgcttt tttgaattcg ctagctaggt cttgaaagga
     7381 gctagctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc
     7441 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag
     7501 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
     7561 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
     7621 gggtttgccg ccagaacaca ggaccggttc tagagcgctg ccaccatgga caagaagtac
     7681 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     7741 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     7801 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     7861 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     7921 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     7981 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     8041 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     8101 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     8161 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     8221 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     8281 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     8341 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     8401 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     8461 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     8521 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     8581 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     8641 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     8701 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     8761 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     8821 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     8881 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     8941 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     9001 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     9061 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     9121 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     9181 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     9241 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     9301 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     9361 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     9421 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     9481 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     9541 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     9601 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     9661 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     9721 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     9781 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     9841 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     9901 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     9961 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
    10021 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
    10081 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
    10141 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
    10201 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
    10261 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
    10321 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
    10381 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
    10441 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
    10501 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
    10561 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
    10621 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
    10681 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
    10741 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
    10801 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
    10861 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
    10921 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
    10981 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
    11041 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
    11101 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
    11161 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
    11221 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
    11281 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
    11341 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
    11401 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
    11461 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
    11521 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
    11581 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
    11641 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
    11701 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
    11761 ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa gaagaaagat
    11821 tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct gaaacaagcc
    11881 ggagatgtcg aagagaatcc tggaccgaaa aagcctgaac tcaccgcgac gtctgtcagc
    11941 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
    12001 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
    12061 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
    12121 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
    12181 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
    12241 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
    12301 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
    12361 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
    12421 aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa
    12481 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
    12541 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
    12601 gacctcccta gcaaactggg gcacaagctt aattaaacgc gttaagtcga caatcaacct
    12661 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
    12721 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
    12781 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
    12841 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
    12901 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
    12961 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
    13021 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
    13081 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
    13141 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
    13201 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
    13261 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
    13321 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
    13381 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
    13441 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
    13501 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc
    13561 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
    13621 gtgccttcct tgaccctgga aggtgccact c
//
