LOCUS       sequence-465550-       13212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knock out of murine ITGA5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13212)
  AUTHORS   Nikolatou K, Sandilands E, Roman-Fernandez A, Cumming EM, Freckmann 
            E, Lilla S, Buetow L, McGarry L, Neilson M, Shaw R, Strachan D, 
            Miller C, Huang DT, McNeish IA, Norman JC, Zanivan S, Bryant DM
  TITLE     PTEN deficiency exposes a requirement for an ARF GTPase module for 
            integrin-dependent invasion in ovarian cancer.
  JOURNAL   EMBO J. 2023 Sep 18;42(18):e113987. doi: 10.15252/embj.2023113987. 
            Epub 2023 Aug 14.
  PUBMED    37577760
REFERENCE   2  (bases 1 to 13212)
  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: "EMBO J."; 
            date: "2023-09-18"; volume: "42(18):e113987. doi"; pages: " 
            10.15252/embj.2023113987. Epub 2023 Aug 14"
COMMENT     Sequence Label: pLentiCRISPR V2 Neo sgITGA5_4
FEATURES             Location/Qualifiers
     source          1..13212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(259..303)
                     /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    488..721
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    1214..1339
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             1386..1566
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(1584..1782)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1609..1629)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1784..2163
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2339..2358
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2433..2537
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2538..2606,2607..3398)
                     /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     complement(2756..2775)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3569..4157
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4058..4077
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4311..4328
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4445..4466
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4481..4486,4487..4504,4505..4511)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4519..4535
                     /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)."
     primer_bind     4524..4546
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4543..4559
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4543..4559
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4608..4729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4619..4638)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4673..4692
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4859..5233)
                     /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"
     promoter        complement(5252..5299)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5347..5676)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5361..5496
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5415..5434)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5661..5681
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5690..6118)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5800..5821)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6012..6031
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6164..6388
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6377..6394
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             6420..6653
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    6725..7313
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6831..6842
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7240..7260
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7329..8120)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7440..7459)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     8050..8069
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(8121..8177)
                     /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             complement(8187..8210)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(8211..8258)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(8259..12362)
                     /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             complement(8259..12362)
                     /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"
     regulatory      12359..12368
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(12387..12598)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(12660..12735)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(12757..13005)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(12815..12834)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(12985..13005)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    13056..13173
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 acgcagtgcc gatccgttca ctaatcgaat ggatctgtct ctgtctctct ctccaccttc
       61 ttcttctatt ccttcgggcc tgtcgggtcc cctcggggtt gggaggtggg tctgaaacga
      121 taatggtgaa tatccctgcc taactctatt cactatagaa agtacagcaa aaactattct
      181 taaacctacc aagcctccta ctatcattat gaataatttt atataccaca gccaatttgt
      241 tatgttaaac caattccaca aacttgccca tttatctaat tccaataatt cttgttcatt
      301 cttttcttgc tggttttgcg attcttcaat taaggagtgt attaagcttg tgtaattgtt
      361 aatttctctg tcccactcca tccaggtcgt gtgattccaa atctgttcca gagatttatt
      421 actccaacta gcattccaag gcacagcagt ggtgcaaatg agttttccag agcaacccca
      481 aatccccagg agctgttgat cctttaggta tctttccaca gccaggattc ttgcctggag
      541 ctgcttgatg ccccagactg tgagttgcaa cagatgctgt tgcgcctcaa tagccctcag
      601 caaattgttc tgctgctgca ctataccaga caataattgt ctggcctgta ccgtcagcgt
      661 cattgacgct gcgcccatag tgcttcctgc tgctcccaag aacccaagga acaaagctcc
      721 tattcccact gctctttttt ctctctgcac cactcttctc tttgccttgg tgggtgctac
      781 tcctaatggt tcaattttta ctactttata tttatataat tcacttctcc aattgtccct
      841 catatctcct cctccaggtc tgaagatcag cggccgcttg ctgtgcggtg gtcttacttt
      901 tgttttgctc ttcctctatc ttgtctaaag cttccttggt gtcttttatc tctatccttt
      961 gatgcacaca atagagggtt gctactgtat tatataatga tctaagttct tctgatcctg
     1021 tctgaaggga tggttgtagc tgtcccagta tttgtctaca gccttctgat gtttctaaca
     1081 ggccaggatt aactgcgaat cgttctagct ccctgcttgc ccatactata tgttttaatt
     1141 tatatttttt ctttccccct ggccttaacc gaattttttc ccatcgcgat ctaattctcc
     1201 cccgcttaat actgacgctc tcgcacccat ctctctcctt ctagcctccg ctagtcaaaa
     1261 tttttggcgt actcaccagt cgccgcccct cgcctcttgc cgtgcgcgct tcagcaagcc
     1321 gagtcctgcg tcgagagagc tcctctggtt tccctttcgc tttcaagtcc ctgttcgggc
     1381 gccactgcta gagattttcc acactgacta aaagggtctg agggatctct agttaccaga
     1441 gtcacacaac agacgggcac acactacttg aagcactcaa ggcaagcttt attgaggctt
     1501 aagcagtggg ttccctagtt agccagagag ctcccaggct cagatctggt ctaaccagag
     1561 agacccagta caggcaaaac gcgctgctta tatagacctc ccaccgtaca cgcctaccgc
     1621 ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt
     1681 gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa
     1741 accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga
     1801 ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat
     1861 gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca
     1921 cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga
     1981 aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt
     2041 cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat
     2101 atgggctatg aactaatgac cccgtaattg attactatta ataactagtc aataatcaat
     2161 gtcaacgcgc gcgtatatct ggcccgtaca tcgcgaagca gcgcaaaacg cctaacccta
     2221 agcagattct tcatgcaatt gtcggtcaag ccttgccttg ttgtagctta aattttgctc
     2281 gcgcactact cagcgacctc caacacacaa gcagggagca gatactggct taactatgcg
     2341 gcatcagagc agattgtact gagagtgcac cataggggat cgggagatct cccgatccgt
     2401 cgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2461 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2521 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2581 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2641 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2701 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     2761 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     2821 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2881 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2941 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3001 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3061 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3121 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3181 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3241 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3301 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3361 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3421 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3481 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3541 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3601 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3661 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     3721 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3781 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3841 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3901 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3961 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4021 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4081 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4141 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4201 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4261 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4321 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     4381 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     4441 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     4501 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     4561 atgattacgc caagctctag ctagaggtcg acggtataca gacatgataa gatacattga
     4621 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     4681 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga
     4741 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     4801 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt agcacgtgtc
     4861 agtcctgctc ctcggccacg aagtgcacgc agttgccggc cgggtcgcgc agggcgaact
     4921 cccgccccca cggctgctcg ccgatctcgg tcatggccgg cccggaggcg tcccggaagt
     4981 tcgtggacac gacctccgac cactcggcgt acagctcgtc caggccgcgc acccacaccc
     5041 aggccagggt gttgtccggc accacctggt cctggaccgc gctgatgaac agggtcacgt
     5101 cgtcccggac cacaccggcg aagtcgtcct ccacgaagtc ccgggagaac ccgagccggt
     5161 cggtccagaa ctcgaccgct ccggcgacgt cgcgcgcggt gagcaccgga acggcactgg
     5221 tcaacttggc catggtttag ttcctcacct tgtcgtatta tactatgccg atatactatg
     5281 ccgatgatta attgtcaaca cgtgctgatc agatccgaaa atggatatac aagctcccgg
     5341 gagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc
     5401 agaggcagag gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg
     5461 agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat
     5521 ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga
     5581 ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
     5641 ggagcctggg gactttccac accctaactg acacacattc cacagaatta attcgcgtta
     5701 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     5761 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     5821 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     5881 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     5941 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     6001 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     6061 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg
     6121 ggataccccc tagagcccca gctggttctt tccgcctcag aagccataga gcccaccgca
     6181 tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca
     6241 ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag
     6301 gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca
     6361 acagatggct ggcaactaga aggcacagtc gaggctgatc agcgggttta aacgggccct
     6421 gctagagatt ttccacactg actaaaaggg tctgagggat ctctagttac cagagtcaca
     6481 caacagacgg gcacacacta cttgaagcac tcaaggcaag ctttattgag gcttaagcag
     6541 tgggttccct agttagccag agagctccca ggctcagatc tggtctaacc agagagaccc
     6601 agtacaagca aaaagcagat cttgtcttcg ttgggagtga attagccctt ccagtccccc
     6661 cttttctttt aaaaagtggc taagatctac agctgccttg taagtcattg gtcttaaagt
     6721 cgacgcgggg aggcggccca aagggagatc cgactcgtct gagggcgaag gcgaagacgc
     6781 ggaagaggcc gcagagccgg cagcaggccg cgggaaggaa ggtccgctgg attgagggcc
     6841 gaagggacgt agcagaagga cgtcccgcgc agaatccagg tggcaacaca ggcgagcagc
     6901 caaggaaagg acgatgattt ccccgacaac accacggaat tgtcagtgcc caacagccga
     6961 gcccctgtcc agcagcgggc aaggcaggcg gcgatgagtt ccgccgtggc aatagggagg
     7021 gggaaagcga aagtcccgga aaggagctga caggtggtgg caatgcccca accagtgggg
     7081 gttgcgtcag caaacacagt gcacaccacg ccacgttgcc tgacaacggg ccacaactcc
     7141 tcataaagag acagcaacca ggatttatac aaggaggaga aaatgaaagc catacgggaa
     7201 gcaatagcat gatacaaagg cattaaagca gcgtatccac atagcgtaaa aggagcaaca
     7261 tagttaagaa taccagtcaa tctttcacaa attttgtaat ccagaggttg attgtcgact
     7321 taacgcgttc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga
     7381 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
     7441 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
     7501 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
     7561 tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg
     7621 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
     7681 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
     7741 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
     7801 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
     7861 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
     7921 gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc ggtcttgaca
     7981 aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
     8041 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
     8101 tgcaatccat cttgttcaat cggtccagga ttctcttcga catctccggc ttgtttcagc
     8161 agagagaagt ttgttgcgcc ggatccctta tcgtcatcgt ctttgtaatc tttcttcttc
     8221 ttagcctgtc cagccttctt tgtggcggca ggtcgcttgt cgcctcccag ctgagacagg
     8281 tcgatccgtg tctcgtacag gccggtgatg ctctggtgga tcagggtggc gtccagcacc
     8341 tctttggtgc tggtgtacct cttccggtcg atggtggtgt caaagtactt gaaggcggca
     8401 ggggctccca gattggtcag ggtaaacagg tggatgatat tctcggcctg ctctctgatg
     8461 ggcttatccc ggtgcttgtt gtaggcggac agcactttgt ccagattagc gtcggccagg
     8521 atcactctct tggagaactc gctgatctgc tcgatgatct cgtccaggta gtgcttgtgc
     8581 tgttccacaa acagctgttt ctgctcatta tcctcggggg agcccttcag cttctcatag
     8641 tggctggcca ggtacaggaa gttcacatat ttggagggca gggccagttc gtttcccttc
     8701 tgcagttcgc cggcagaggc cagcattctc ttccggccgt tttccagctc gaacagggag
     8761 tacttaggca gcttgatgat caggtccttt ttcacttctt tgtagccctt ggcttccaga
     8821 aagtcgatgg gattcttctc gaagctgctt ctttccatga tggtgatccc cagcagctct
     8881 ttcacactct tcagtttctt ggacttgccc ttttccactt tggccaccac cagcacagaa
     8941 taggccacgg tggggctgtc gaagccgccg tacttcttag ggtcccagtc cttctttctg
     9001 gcgatcagct tatcgctgtt cctcttgggc aggatagact ctttgctgaa gccgcctgtc
     9061 tgcacctcgg tctttttcac gatattcact tggggcatgc tcagcacttt ccgcacggtg
     9121 gcaaaatccc ggcccttatc ccacacgatc tccccggttt cgccgtttgt ctcgatcaga
     9181 ggccgcttcc ggatctcgcc gttggccagg gtaatctcgg tcttgaaaaa gttcatgatg
     9241 ttgctgtaga agaagtactt ggcggtagcc ttgccgattt cctgctcgct cttggcgatc
     9301 atcttccgca cgtcgtacac cttgtagtcg ccgtacacga actcgctttc cagcttaggg
     9361 tactttttga tcagggcggt tcccacgacg gcgttcaggt aggcgtcgtg ggcgtggtgg
     9421 tagttgttga tctcgcgcac tttgtaaaac tggaaatcct tccggaaatc ggacaccagc
     9481 ttggacttca gggtgatcac tttcacttcc cggatcagct tgtcattctc gtcgtactta
     9541 gtgttcatcc gggagtccag gatctgtgcc acgtgctttg tgatctgccg ggtttccacc
     9601 agctgtctct tgatgaagcc ggccttatcc agttcgctca ggccgcctct ctcggccttg
     9661 gtcagattgt cgaactttct ctgggtaatc agcttggcgt tcagcagctg ccgccagtag
     9721 ttcttcatct tcttcacgac ctcttcggag ggcacgttgt cgctcttgcc ccggttcttg
     9781 tcgcttctgg tcagcacctt gttgtcgatg gagtcgtcct tcagaaagct ctgaggcacg
     9841 atatggtcca catcgtagtc ggacagccgg ttgatgtcca gttcctggtc cacgtacata
     9901 tcccgcccat tctgcaggta gtacaggtac agcttctcgt tctgcagctg ggtgttttcc
     9961 acggggtgtt ctttcaggat ctggctgccc agctctttga tgccctcttc gatccgcttc
    10021 attctctcgc ggctgttctt ctgtcccttc tgggtggtct ggttctctct ggccatttcg
    10081 atcacgatgt tctcgggctt gtgccggccc atcactttca cgagctcgtc caccaccttc
    10141 actgtctgca ggatgccctt cttaatggcg gggctgccgg ccagattggc aatgtgctcg
    10201 tgcaggctat cgccctggcc ggacacctgg gctttctgga tgtcctcttt aaaggtcagg
    10261 ctgtcgtcgt ggatcagctg catgaagttt ctgttggcga agccgtcgga cttcaggaaa
    10321 tccaggattg tcttgccgga ctgcttgtcc cggatgccgt tgatcagctt ccggctcagc
    10381 ctgccccagc cggtgtatct ccgccgcttc agctgcttca tcactttgtc gtcgaacagg
    10441 tgggcatagg ttttcagccg ttcctcgatc atctctctgt cctcaaacag tgtcagggtc
    10501 agcacgatat cttccagaat gtcctcgttt tcctcattgt ccaggaagtc cttgtccttg
    10561 ataattttca gcagatcgtg gtatgtgccc agggaggcgt tgaaccgatc ttccacgccg
    10621 gagatttcca cggagtcgaa gcactcgatt ttcttgaagt agtcctcttt cagctgcttc
    10681 acggtcactt tccggttggt cttgaacagc aggtccacga tggccttttt ctgctcgccg
    10741 ctcaggaagg cgggctttct cattccctcg gtcacgtatt tcactttggt cagctcgtta
    10801 tacacggtga agtactcgta cagcaggctg tgcttgggca gcaccttctc gttgggcagg
    10861 ttcttatcga agttggtcat ccgctcgatg aagctctggg cggaagcgcc cttgtccacc
    10921 acttcctcga agttccaggg ggtgatggtt tcctcgctct ttctggtcat ccaggcgaat
    10981 ctgctgtttc ccctggccag agggcccacg tagtagggga tgcggaaggt caggatcttc
    11041 tcgatctttt cccggttgtc cttcaggaat gggtaaaaat cttcctgccg ccgcagaatg
    11101 gcgtgcagct ctcccaggtg gatctggtgg gggatgctgc cgttgtcgaa ggtccgctgc
    11161 ttccgcagca ggtcctctct gttcagcttc acgagcagtt cctcggtgcc gtccatcttt
    11221 tccaggatgg gcttgatgaa cttgtagaac tcttcctggc tggctccgcc gtcaatgtag
    11281 ccggcgtagc cgttcttgct ctggtcgaag aaaatctctt tgtacttctc aggcagctgc
    11341 tgccgcacga gagctttcag cagggtcagg tcctggtggt gctcgtcgta tctcttgatc
    11401 atagaggcgc tcaggggggc cttggtgatc tcggtgttca ctctcaggat gtcgctcagc
    11461 aggatggcgt cggacaggtt cttggcggcc agaaacaggt cggcgtactg gtcgccgatc
    11521 tgggccagca ggttgtccag gtcgtcgtcg taggtgtcct tgctcagctg cagtttggca
    11581 tcctcggcca ggtcgaagtt gctcttgaag ttgggggtca ggcccaggct cagggcaatc
    11641 aggtttccga acaggccatt cttcttctcg ccgggcagct gggcgatcag attttccagc
    11701 cgtctgctct tgctcagtct ggcagacagg atggccttgg cgtccacgcc gctggcgttg
    11761 atggggtttt cctcgaacag ctggttgtag gtctgcacca gctggatgaa cagcttgtcc
    11821 acgtcgctgt tgtcggggtt caggtcgccc tcgatcagga agtggccccg gaacttgatc
    11881 atgtgggcca gggccagata gatcagccgc aggtcggcct tgtcggtgct gtccaccagt
    11941 ttctttctca ggtggtagat ggtggggtac ttctcgtggt aggccacctc gtccacgatg
    12001 ttgccgaaga tggggtgccg ctcgtgcttc ttatcctctt ccaccaggaa ggactcttcc
    12061 agtctgtgga agaagctgtc gtccaccttg gccatctcgt tgctgaagat ctcttgcaga
    12121 tagcagatcc ggttcttccg tctggtgtat cttcttctgg cggttctctt cagccgggtg
    12181 gcctcggctg tttcgccgct gtcgaacagc agggctccga tcaggttctt cttgatgctg
    12241 tgccggtcgg tgttgcccag caccttgaat ttcttgctgg gcaccttgta ctcgtcggtg
    12301 atcacggccc agcccacaga gttggtgccg atgtccaggc cgatgctgta cttcttgtcc
    12361 atggtggcag cgctctagaa ccggtcctgt gttctggcgg caaacccgtt gcgaaaaaga
    12421 acgttcacgg cgactactgc acttatatac ggttctcccc caccctcggg aaaaaggcgg
    12481 agccagtaca cgacatcact ttcccagttt accccgcgcc accttctcta ggcaccggat
    12541 caattgccga cccctccccc caacttctcg gggactgtgg gcgatgtgcg ctctgcccac
    12601 tgacgggcac cggagccaat tcccactcct ttcaagacct agctagcgaa ttcaaaaaag
    12661 caccgactcg gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat
    12721 ttctagctct aaaaccgtct aggttgaagc ccccacggtg tttcgtcctt tccacaagat
    12781 atataaagcc aagaaatcga aatactttca agttacggta agcatatgat agtccatttt
    12841 aaaacataat tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt
    12901 ttgtactaat atctttgtgt ttacagtcaa attaattcta attatctctc taacagcctt
    12961 gtatcgtata tgcaaatatg aaggaatcat gggaaatagg ccctcggtac cttaattaac
    13021 caaactggat ctctgctgtc cctgtaataa acccgaaaat tttgaatttt tgtaatttgt
    13081 ttttgtaatt ctttagtttg tatgtctgtt gctattatgt ctactattct ttcccctgca
    13141 ctgtaccccc caatcccccc ttttctttta aaattgtgga tgaatactgc catttgtctg
    13201 cagaattggc gc
//
