LOCUS       sequence-362647-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets MAP2K2 
            gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11691)
  AUTHORS   Hu M, Lei XY, Larson JD, McAlonis M, Ford K, McDonald D, Mach K, 
            Rusert JM, Wechsler-Reya RJ, Mali P
  TITLE     Integrated genome and tissue engineering enables screening of cancer
            vulnerabilities in physiologically relevant perfusable ex vivo 
            cultures.
  JOURNAL   Biomaterials. 2022 Jan;280:121276. doi: 
            10.1016/j.biomaterials.2021.121276. Epub 2021 Dec 2.
  PUBMED    34890975
REFERENCE   2  (bases 1 to 11691)
  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: 
            "Biomaterials."; date: "2022-01"; volume: "280:121276. doi"; pages: 
            " 10.1016/j.biomaterials.2021.121276. Epub 2021 Dec 2"
COMMENT     Sequence Label: CROP_C9_Puro_MAP2K2
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..31
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     15..31
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     50..70
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        52..70
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        98..324
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             325..505
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    552..677
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1170..1403
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1588..1632
                     /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    1813..1930
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2119..2330
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2349..2358
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2355..6458
                     /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             2355..6458
                     /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             6459..6506
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6507..6530
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6540..6596
                     /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             6597..7196
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6597..6616)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7093..7113
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7212..7800
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7265..7285)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7683..7694)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7956..8204
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7956..7976
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8127..8146
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8226..8301
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8354..8534
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8612..8733
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8649..8668)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8703..8722
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8773..8908
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8928..8947)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8929..8947)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8957..8974)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8957..8973)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8966..8988)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9115..9570
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9202..9221)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9412..9433
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9596..9700
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9701..9769,9770..10561)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(9919..9938)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10732..11320
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11221..11240
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11474..11491
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11608..11629
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11644..11649,11650..11667,11668..11674)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(11682..11691,1..7)
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     join(11687..11691,1..18)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
       61 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
      121 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
      181 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
      241 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
      301 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
      361 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
      421 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
      481 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
      541 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
      601 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
      661 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
      721 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
      781 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
      841 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
      901 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
      961 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1021 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     1081 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     1141 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     1201 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     1261 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     1321 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     1381 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     1441 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     1501 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     1561 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     1621 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     1681 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     1741 aatagagtta ggcagggata ttcaccatta tcgtttcaga atcttgagac aaatggcagt
     1801 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     1861 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     1921 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgaggg
     1981 ggcccgggtg caaagatgga taaagtttta aacagagagg aatctttgca gctaatggac
     2041 cttctaggtc ttgaaaggag tgggaattgg ctccgtaggt cttgaaagga gtgggaattg
     2101 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     2161 gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     2221 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     2281 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gaccggttct
     2341 agagcgctgc caccatggac aagaagtaca gcatcggcct ggacatcggc accaactctg
     2401 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     2461 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     2521 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     2581 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     2641 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     2701 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     2761 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     2821 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     2881 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     2941 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     3001 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     3061 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     3121 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     3181 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     3241 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     3301 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     3361 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     3421 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     3481 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     3541 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     3601 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     3661 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     3721 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     3781 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     3841 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     3901 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     3961 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     4021 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     4081 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     4141 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     4201 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     4261 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     4321 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     4381 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     4441 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     4501 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     4561 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     4621 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     4681 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     4741 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     4801 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     4861 acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     4921 tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     4981 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     5041 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     5101 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     5161 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     5221 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     5281 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     5341 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     5401 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     5461 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     5521 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
     5581 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
     5641 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     5701 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     5761 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     5821 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
     5881 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     5941 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     6001 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     6061 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     6121 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
     6181 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
     6241 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
     6301 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     6361 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     6421 acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga
     6481 aggctggaca ggctaagaag aagaaagatt acaaagacga tgacgataag ggatccggcg
     6541 caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgatga
     6601 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     6661 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     6721 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     6781 gcaaggtgtg ggtcgcggac gacggcgccg ccgtggcggt ctggaccacg ccggagagcg
     6841 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     6901 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     6961 cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg
     7021 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     7081 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     7141 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc
     7201 gttaagtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     7261 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     7321 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     7381 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     7441 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     7501 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     7561 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     7621 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     7681 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     7741 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     7801 gtcgacttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     7861 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatttca ccattatcgt
     7921 ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc atgattcctt
     7981 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     8041 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     8101 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     8161 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggtagg gttgatggtg
     8221 agcgcgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     8281 aaagtggcac cgagtcggtg cttttttaag cttggcgtaa ctagatcttg agacactgct
     8341 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     8401 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     8461 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     8521 gaaaatctct agcagtacgt atagtagttc atgtcatctt attattcagt atttataact
     8581 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
     8641 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     8701 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
     8761 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     8821 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     8881 aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt
     8941 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     9001 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     9061 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc
     9121 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     9181 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     9241 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     9301 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     9361 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     9421 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     9481 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     9541 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     9601 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     9661 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     9721 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     9781 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     9841 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     9901 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     9961 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
    10021 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
    10081 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
    10141 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
    10201 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
    10261 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
    10321 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
    10381 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
    10441 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
    10501 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
    10561 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
    10621 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
    10681 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
    10741 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
    10801 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
    10861 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
    10921 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
    10981 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
    11041 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
    11101 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
    11161 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
    11221 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
    11281 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
    11341 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
    11401 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
    11461 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
    11521 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
    11581 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
    11641 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg g
//
