LOCUS       sequence-431413-       13097 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one, knockout, delivers Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13097)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 13097)
  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: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: pRDA_734
FEATURES             Location/Qualifiers
     source          1..13097
                     /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        2615..2863
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2615..2635
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2786..2805
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2983..3194
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3199..3208
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3244..3264
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3271..7371
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             3271..7371
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7378..7425
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7432..7512
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             7519..7575
                     /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             7576..8169
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     8069..8089
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8189..8777
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8242..8262)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8660..8671)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8849..9082
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9108..9125)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9114..9338
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9384..9812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9471..9490)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9681..9702
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9821..9841)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9826..10155
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10006..10141
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10068..10087
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10203..10250
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10269..10643
                     /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    10773..10894
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10810..10829)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10864..10883
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10943..10959)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10943..10959)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10956..10978)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10967..10983
                     /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(10991..10997,10998..11015,11016..11021))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11036..11057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11174..11191)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11345..11933)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11425..11444)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12104..12895,12896..12964))
                     /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     12727..12746
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12965..13069)
                     /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 aggtaccggg cagagagggc ctatttccca tgattccttc
     2641 atatttgcat atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa
     2701 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     2761 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     2821 cgatttcttg gctttatata tcttgtggaa aggacgaaac accggagacg cttttttcgt
     2881 ctcagtttaa gagctatgct ggaaacagca tagcaagttt aaataaggct agtccgttat
     2941 caacttgaaa aagtggcacc gagtcggtgc ttttttgaac acgggcagag cgcacatcgc
     3001 ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt
     3061 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     3121 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     3181 ccgccagaac acagaggagc caccatggcc aaacggacag ccgacggaag cgagttcgag
     3241 tcaccaaaga agaagcggaa agtcggcagc gacaagaagt acagcatcgg cctggacatc
     3301 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     3361 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     3421 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     3481 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     3541 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     3601 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     3661 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     3721 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     3781 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     3841 tacaaccagc tgttcgagga aaaccccatc aacgctagcg gcgtggacgc caaggccatc
     3901 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     3961 gagaagaaga atggcctgtt cggcaacctg attgccctga gcctgggcct gacccccaac
     4021 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     4081 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     4141 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     4201 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     4261 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     4321 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     4381 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     4441 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     4501 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     4561 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     4621 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     4681 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     4741 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     4801 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     4861 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     4921 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     4981 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     5041 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     5101 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     5161 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     5221 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     5281 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     5341 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     5401 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     5461 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     5521 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     5581 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     5641 ggcagccaga tcctgaaaga acacccagtg gaaaacaccc agctgcagaa cgagaagctg
     5701 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     5761 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     5821 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     5881 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     5941 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     6001 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     6061 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     6121 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     6181 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     6241 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     6301 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     6361 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     6421 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     6481 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     6541 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     6601 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     6661 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     6721 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     6781 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     6841 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     6901 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     6961 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     7021 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     7081 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     7141 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     7201 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     7261 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     7321 atcaccggcc tgtacgagac acgaatcgat ttgtctcagc tgggaggcga tgggtccaaa
     7381 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc ctacccatac
     7441 gatgttccag attacgctta tccctacgac gtgcctgatt atgcataccc atatgatgtc
     7501 cccgactatg ccggaagcgc aacaaacttc tctctgctga aacaagccgg agatgtcgaa
     7561 gagaatcctg gaccgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     7621 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     7681 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     7741 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     7801 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     7861 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     7921 cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc
     7981 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     8041 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     8101 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     8161 cccggtgccg gatcctgata aattcgaaaa tcaacctctg gattacaaaa tttgtgaaag
     8221 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     8281 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     8341 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     8401 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     8461 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     8521 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     8581 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     8641 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     8701 gccggctctg cggcctcttc cgcgtcctcg ccttcgccct cagacgagtc ggatctccct
     8761 ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt
     8821 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagataa
     8881 gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     8941 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     9001 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     9061 tcagtgtgga aaatctctag cagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     9121 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     9181 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     9241 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     9301 taatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     9361 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     9421 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     9481 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     9541 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     9601 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     9661 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     9721 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     9781 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     9841 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     9901 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     9961 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
    10021 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
    10081 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
    10141 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac
    10201 gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga
    10261 actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg
    10321 gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact
    10381 tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg
    10441 tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt
    10501 ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg
    10561 gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact
    10621 tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc gccgccttct
    10681 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
    10741 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
    10801 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
    10861 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
    10921 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
    10981 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
    11041 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
    11101 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
    11161 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
    11221 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
    11281 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
    11341 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
    11401 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
    11461 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
    11521 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
    11581 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
    11641 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
    11701 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
    11761 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
    11821 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
    11881 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
    11941 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
    12001 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
    12061 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
    12121 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
    12181 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
    12241 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
    12301 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
    12361 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
    12421 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
    12481 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
    12541 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
    12601 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
    12661 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
    12721 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
    12781 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
    12841 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
    12901 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
    12961 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
    13021 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
    13081 gaaaagtgcc acctgac
//
