LOCUS       sequence-428816-       13902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression vector for an inducible Cas9-P2A-Venus with 
            two sgRNA sequences against human non-coding DNA region of 
            chromosome 1 (negative control).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13902)
  TITLE     Selectable lentivectors delivering dual gRNAs and destabilized Cas9
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13902)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pDual_dsCas9_Venus_hs_NC_chr1
FEATURES             Location/Qualifiers
     source          1..13902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2608..2848
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     misc_RNA        2879..2954
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3045..3285
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     misc_RNA        3313..3388
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3450..3661
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3675..3684
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3681..3683,3684..4004)
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKPE"
     CDS             4014..8117
                     /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             4014..8117
                     /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             8118..8165
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8166..8189
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             8199..8255
                     /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             join(8259..8261,8262..8264,8265..8978)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8565..8584)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8912..8933
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    8994..9582
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9047..9067)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9465..9476)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9654..9887
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9913..9930)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9919..10143
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10189..10617
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10276..10295)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10486..10507
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10626..10646)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10631..10960
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10811..10946
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10873..10892
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11008..11055
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11074..11448
                     /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    11578..11699
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11615..11634)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11669..11688
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11748..11764)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11748..11764)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11761..11783)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11772..11788
                     /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(11796..11802,11803..11820,11821..11826))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11841..11862
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11979..11996)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12150..12738)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12230..12249)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12909..13700,13701..13769))
                     /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     13532..13551
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13770..13874)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacctgcatt gttgttatag tgtgagtggt
     2881 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     2941 caccgagtcg gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc
     3001 cgtttttagc gcgtgcgcca attctgcaga caaatggctc tagagagggc ctatttccca
     3061 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
     3121 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     3181 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     3241 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggaccgtct ctagaaatca
     3301 tcctagactt aagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     3361 acttgaaaaa gtggcaccga gtcggtgctt ttttgaattc gctagctagg tcttgaaagg
     3421 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
     3481 gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa
     3541 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
     3601 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
     3661 ggaccggttc tagagccacc atgggagtgc aggtggaaac catctcccca ggtgacgggc
     3721 gcaccttccc caagcgcggc cagacctgtg tggtgcacta caccgggatg cttgaagatg
     3781 gaaagaaagt cgattcctcc cgggacagaa acaagccctt taagtttatg ctaggcaagc
     3841 aggaggtgat ccgaggctgg gaagaagggg ttgcccagat gagtgtgggt cagagagcca
     3901 aactgactat atctccagat tatgcctatg gtgccactgg gcacccaggc atcatcccac
     3961 cacatgccac tctcgtcttc gatgtggagc ttctaaaacc ggaaccggct agcatggaca
     4021 agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg
     4081 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
     4141 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc
     4201 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
     4261 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
     4321 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
     4381 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
     4441 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
     4501 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     4561 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     4621 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg
     4681 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg
     4741 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     4801 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     4861 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     4921 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     4981 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     5041 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     5101 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     5161 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc
     5221 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     5281 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     5341 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     5401 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     5461 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     5521 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg
     5581 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     5641 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     5701 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     5761 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     5821 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     5881 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     5941 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     6001 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     6061 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     6121 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     6181 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     6241 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     6301 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     6361 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     6421 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     6481 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc
     6541 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga
     6601 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     6661 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg
     6721 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     6781 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     6841 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     6901 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     6961 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     7021 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     7081 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     7141 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     7201 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg
     7261 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     7321 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa
     7381 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     7441 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     7501 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     7561 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     7621 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     7681 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     7741 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     7801 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     7861 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca
     7921 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     7981 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     8041 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     8101 ctcagctggg aggcgacaag cgacctgccg ccacaaagaa ggctggacag gctaagaaga
     8161 agaaagatta caaagacgat gacgataagg gatccggagc tactaacttc agcctgctga
     8221 agcaggctgg ggacgtggag gagaaccctg gacctaggat ggtgagcaag ggcgaggagc
     8281 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     8341 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagctga
     8401 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgggctacg
     8461 gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     8521 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     8581 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     8641 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     8701 gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     8761 tccgccacaa catcgaggac ggcggcgtgc agctcgccga ccactaccag cagaacaccc
     8821 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc
     8881 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     8941 ccgggatcac tctcggcatg gacgagctgt acaagtaagg atcctaagtc gacaatcaac
     9001 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     9061 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     9121 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     9181 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     9241 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     9301 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     9361 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg
     9421 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     9481 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     9541 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat
     9601 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     9661 gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt
     9721 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     9781 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     9841 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa
     9901 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     9961 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
    10021 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
    10081 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
    10141 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
    10201 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
    10261 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
    10321 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
    10381 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
    10441 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
    10501 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
    10561 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
    10621 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
    10681 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
    10741 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
    10801 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
    10861 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
    10921 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
    10981 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
    11041 atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg
    11101 gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc
    11161 tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc
    11221 atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc
    11281 ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc
    11341 tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc
    11401 gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga
    11461 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
    11521 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
    11581 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
    11641 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
    11701 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
    11761 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
    11821 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
    11881 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
    11941 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
    12001 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
    12061 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
    12121 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
    12181 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
    12241 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
    12301 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
    12361 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
    12421 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
    12481 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
    12541 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
    12601 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
    12661 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
    12721 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
    12781 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
    12841 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
    12901 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
    12961 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
    13021 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
    13081 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
    13141 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
    13201 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
    13261 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
    13321 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
    13381 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
    13441 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
    13501 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
    13561 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
    13621 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
    13681 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
    13741 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
    13801 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
    13861 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ac
//
