LOCUS       sequence-451532-       13401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of spCas9 and sgRNA targeting SIGIRR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13401)
  AUTHORS   Castagnino P, Kim HW, Lam LKM, Basu D, White EA
  TITLE     Systematic Analysis of IL-1 Cytokine Signaling Suppression by HPV16 
            Oncoproteins.
  JOURNAL   J Virol. 2022 Nov 7:e0132622. doi: 10.1128/jvi.01326-22.
  PUBMED    36342298
REFERENCE   2  (bases 1 to 13401)
  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: "J Virol. 
            2022 Nov 7:e0132622. doi: 10.1128/jvi.01326-22."
COMMENT     Sequence Label: p8270 LentiCRISPR v2 hygro sgSIGIRR-3
FEATURES             Location/Qualifiers
     source          1..13401
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1981..2000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2176..2555
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2557..2755
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2710..2730
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2773..2953
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3000..3125
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3618..3851
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4036..4080
                     /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    4378..4495
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4546..4794
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4546..4566
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4717..4736
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4816..4891
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4953..5164
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5183..5192
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5189..9292
                     /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             5189..9292
                     /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             9293..9340
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9341..9364
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             9374..9430
                     /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"
     misc_feature    10427..11015
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10480..10500)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10898..10909)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             11087..11320
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(11346..11363)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11352..11576
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11622..12050
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11709..11728)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11919..11940
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12059..12079)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        12064..12393
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      12244..12379
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12306..12325
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12441..12488
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12507..12881
                     /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    13011..13132
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13048..13067)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13102..13121
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(13181..13197)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13181..13197)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13194..13216)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13205..13221
                     /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(13229..13235,13236..13253,13254..13259))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13274..13295
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     1981 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     2041 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     2101 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     2161 atatacgcgc gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     2221 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     2281 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     2341 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     2401 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     2461 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     2521 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     2581 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     2641 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     2701 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt
     2761 tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     2821 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     2881 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     2941 aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc
     3001 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac
     3061 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga
     3121 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag
     3181 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
     3241 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc
     3301 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa
     3361 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga
     3421 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga
     3481 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     3541 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     3601 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     3661 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     3721 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     3781 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     3841 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     3901 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     3961 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     4021 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     4081 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     4141 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     4201 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg
     4261 cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg
     4321 aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt
     4381 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     4441 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     4501 ttattacagg gacagcagag atccagtttg gttaattaag gtaccgaggg cctatttccc
     4561 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     4621 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     4681 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     4741 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggggcc
     4801 cgggatacca gcgcggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     4861 tcaacttgaa aaagtggcac cgagtcggtg cttttttgaa ttcgctagct aggtcttgaa
     4921 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     4981 cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt
     5041 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     5101 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     5161 acaggaccgg ttctagagcg ctgccaccat ggacaagaag tacagcatcg gcctggacat
     5221 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     5281 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     5341 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     5401 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     5461 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     5521 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     5581 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     5641 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     5701 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     5761 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     5821 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     5881 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     5941 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     6001 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     6061 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     6121 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     6181 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     6241 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     6301 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     6361 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     6421 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     6481 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     6541 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     6601 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     6661 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     6721 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     6781 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     6841 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     6901 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     6961 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     7021 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     7081 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     7141 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     7201 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     7261 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     7321 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     7381 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     7441 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     7501 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     7561 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     7621 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     7681 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     7741 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     7801 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     7861 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     7921 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     7981 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     8041 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     8101 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     8161 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     8221 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     8281 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     8341 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     8401 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     8461 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     8521 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     8581 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     8641 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     8701 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     8761 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     8821 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     8881 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     8941 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     9001 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     9061 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     9121 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     9181 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     9241 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaagcgacc
     9301 tgccgccaca aagaaggctg gacaggctaa gaagaagaaa gattacaaag acgatgacga
     9361 taagggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa
     9421 tcctggaccg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa
     9481 gttcgacagc gtgtccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag
     9541 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta
     9601 caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct
     9661 tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt
     9721 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc
     9781 catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc
     9841 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca
     9901 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct
     9961 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga
    10021 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag
    10081 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg
    10141 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg
    10201 atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt
    10261 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg
    10321 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac
    10381 cgatggctgt gtagaagtac ttcgccgata gacgcgttaa gtcgacaatc aacctctgga
    10441 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
    10501 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
    10561 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
    10621 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
    10681 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
    10741 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
    10801 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
    10861 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
    10921 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
    10981 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
    11041 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
    11101 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
    11161 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
    11221 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
    11281 tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg
    11341 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
    11401 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
    11461 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
    11521 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
    11581 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
    11641 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
    11701 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
    11761 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
    11821 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
    11881 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
    11941 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
    12001 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
    12061 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
    12121 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
    12181 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
    12241 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
    12301 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
    12361 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
    12421 ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat
    12481 aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca
    12541 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
    12601 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
    12661 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
    12721 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
    12781 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
    12841 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg
    12901 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
    12961 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
    13021 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
    13081 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
    13141 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
    13201 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
    13261 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
    13321 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
    13381 gcggtttgcg tattgggcgc t
//
