LOCUS       sequence-362646-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets MAP2K1 
            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_MAP2K1
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..25
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9..25
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     44..64
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        46..64
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        92..318
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             319..499
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    546..671
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1164..1397
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1582..1626
                     /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    1807..1924
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2113..2324
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2343..2352
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2349..6452
                     /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             2349..6452
                     /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             6453..6500
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6501..6524
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6534..6590
                     /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             6591..7190
                     /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(6591..6610)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7087..7107
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7206..7794
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7259..7279)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7677..7688)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7950..8198
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7950..7970
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8121..8140
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8220..8295
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8348..8528
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8606..8727
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8643..8662)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8697..8716
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8767..8902
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8922..8941)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8923..8941)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8951..8968)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8951..8967)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8960..8982)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9109..9564
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9196..9215)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9406..9427
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9590..9694
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9695..9763,9764..10555)
                     /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(9913..9932)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10726..11314
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11215..11234
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11468..11485
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11602..11623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11638..11643,11644..11661,11662..11668)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(11676..11691,1)
                     /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(11681..11691,1..12)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
       61 aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct
      121 tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca
      181 tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt
      241 ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc
      301 ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
      361 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
      421 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
      481 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
      541 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
      601 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
      661 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
      721 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
      781 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
      841 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
      901 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
      961 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc
     1021 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     1081 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     1141 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     1201 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     1261 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     1321 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     1381 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     1441 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     1501 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     1561 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     1621 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     1681 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     1741 gttaggcagg gatattcacc attatcgttt cagaatcttg agacaaatgg cagtattcat
     1801 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     1861 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     1921 ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg agggggcccg
     1981 ggtgcaaaga tggataaagt tttaaacaga gaggaatctt tgcagctaat ggaccttcta
     2041 ggtcttgaaa ggagtgggaa ttggctccgt aggtcttgaa aggagtggga attggctccg
     2101 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
     2161 tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
     2221 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
     2281 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggaccgg ttctagagcg
     2341 ctgccaccat ggacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct
     2401 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
     2461 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa
     2521 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
     2581 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
     2641 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
     2701 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
     2761 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
     2821 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     2881 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     2941 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     3001 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     3061 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     3121 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     3181 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     3241 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     3301 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     3361 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     3421 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     3481 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     3541 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     3601 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     3661 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     3721 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     3781 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact
     3841 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     3901 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     3961 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga
     4021 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     4081 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     4141 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     4201 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     4261 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     4321 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
     4381 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
     4441 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     4501 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     4561 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     4621 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     4681 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     4741 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     4801 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     4861 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
     4921 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     4981 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
     5041 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     5101 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     5161 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
     5221 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     5281 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
     5341 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     5401 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     5461 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     5521 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
     5581 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
     5641 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
     5701 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     5761 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     5821 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     5881 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     5941 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     6001 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     6061 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     6121 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     6181 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     6241 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     6301 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     6361 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     6421 cacggatcga cctgtctcag ctgggaggcg acaagcgacc tgccgccaca aagaaggctg
     6481 gacaggctaa gaagaagaaa gattacaaag acgatgacga taagggatcc ggcgcaacaa
     6541 acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg atgaccgagt
     6601 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     6661 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     6721 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     6781 tgtgggtcgc ggacgacggc gccgccgtgg cggtctggac cacgccggag agcgtcgaag
     6841 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     6901 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     6961 tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     7021 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     7081 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     7141 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag
     7201 tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     7261 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     7321 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     7381 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     7441 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     7501 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     7561 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     7621 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     7681 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     7741 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac
     7801 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     7861 gggactggaa gggctaattc actcccaacg aagacaagat ttcaccatta tcgtttcaga
     7921 cccacctccc aaccccgagg ggacccagag agggcctatt tcccatgatt ccttcatatt
     7981 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
     8041 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     8101 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     8161 tcttggcttt atatatcttg tggaaaggac gaaacaccgt cttacccaga agcagaaggg
     8221 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     8281 gcaccgagtc ggtgcttttt taagcttggc gtaactagat cttgagacac tgctttttgc
     8341 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     8401 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     8461 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     8521 ctctagcagt acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
     8581 gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata
     8641 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     8701 tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact
     8761 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     8821 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     8881 tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt
     8941 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     9001 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     9061 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     9121 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     9181 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     9241 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     9301 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     9361 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     9421 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     9481 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     9541 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     9601 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     9661 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     9721 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     9781 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     9841 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     9901 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     9961 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
    10021 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
    10081 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
    10141 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
    10201 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
    10261 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
    10321 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
    10381 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
    10441 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
    10501 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
    10561 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
    10621 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
    10681 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
    10741 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
    10801 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
    10861 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
    10921 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
    10981 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
    11041 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
    11101 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
    11161 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
    11221 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
    11281 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
    11341 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
    11401 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
    11461 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
    11521 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
    11581 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
    11641 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac a
//
