LOCUS       sequence-362617-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets DHFR 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_DHFR
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    14..30
                     /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     19..41
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     38..54
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     38..54
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     73..93
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        75..93
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        121..347
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             348..528
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    575..700
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1193..1426
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1611..1655
                     /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    1836..1953
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2142..2353
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2372..2381
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2378..6481
                     /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             2378..6481
                     /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             6482..6529
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6530..6553
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6563..6619
                     /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             6620..7219
                     /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(6620..6639)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7116..7136
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7235..7823
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7288..7308)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7706..7717)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7979..8227
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7979..7999
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8150..8169
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8249..8324
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8377..8557
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8635..8756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8672..8691)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8726..8745
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8796..8931
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8951..8970)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8952..8970)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8980..8997)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8980..8996)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8989..9011)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9138..9593
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9225..9244)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9435..9456
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9619..9723
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9724..9792,9793..10584)
                     /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(9942..9961)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10755..11343
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11244..11263
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11497..11514
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11631..11652
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11667..11672,11673..11690,11691,1..6)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
       61 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa
      121 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
      181 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      241 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      301 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      361 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      421 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      481 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      541 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      601 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      661 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      721 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      781 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      841 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      901 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      961 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
     1021 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     1081 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1141 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1201 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1261 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1321 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1381 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1441 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1501 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1561 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1621 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1681 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1741 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1801 agaatcttga gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     1861 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     1921 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     1981 ccactttggc gccggctcga gggggcccgg gtgcaaagat ggataaagtt ttaaacagag
     2041 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccgta
     2101 ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc
     2161 cacagtcccc gagaagttgg ggggaggggt cggcaattga tccggtgcct agagaaggtg
     2221 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     2281 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     2341 cgccagaaca caggaccggt tctagagcgc tgccaccatg gacaagaagt acagcatcgg
     2401 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     2461 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     2521 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     2581 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     2641 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     2701 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     2761 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     2821 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     2881 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     2941 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     3001 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     3061 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     3121 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     3181 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     3241 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     3301 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     3361 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     3421 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     3481 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     3541 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     3601 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     3661 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     3721 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     3781 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     3841 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     3901 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     3961 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     4021 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     4081 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     4141 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     4201 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     4261 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     4321 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     4381 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     4441 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     4501 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     4561 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     4621 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     4681 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     4741 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     4801 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     4861 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
     4921 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     4981 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     5041 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     5101 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     5161 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     5221 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     5281 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     5341 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
     5401 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     5461 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     5521 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
     5581 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     5641 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     5701 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     5761 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
     5821 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     5881 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     5941 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     6001 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
     6061 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     6121 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     6181 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     6241 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     6301 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     6361 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
     6421 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
     6481 caagcgacct gccgccacaa agaaggctgg acaggctaag aagaagaaag attacaaaga
     6541 cgatgacgat aagggatccg gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt
     6601 cgaagagaat cctggaccga tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     6661 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     6721 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     6781 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgccgtggc
     6841 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     6901 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     6961 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct cgcccgacca
     7021 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     7081 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     7141 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     7201 ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa cctctggatt acaaaatttg
     7261 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     7321 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     7381 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     7441 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     7501 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     7561 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     7621 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     7681 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     7741 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     7801 ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta
     7861 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     7921 agacaagatt tcaccattat cgtttcagac ccacctccca accccgaggg gacccagaga
     7981 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     8041 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     8101 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     8161 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     8221 aaacaccgcg gcccggcaga tacctgaggt tttagagcta gaaatagcaa gttaaaataa
     8281 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt aagcttggcg
     8341 taactagatc ttgagacact gctttttgct tgtactgggt ctctctggtt agaccagatc
     8401 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     8461 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     8521 ctcagaccct tttagtcagt gtggaaaatc tctagcagta cgtatagtag ttcatgtcat
     8581 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg
     8641 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     8701 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     8761 gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     8821 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     8881 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt
     8941 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     9001 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     9061 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     9121 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     9181 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     9241 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     9301 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     9361 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     9421 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     9481 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     9541 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     9601 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     9661 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     9721 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     9781 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     9841 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     9901 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     9961 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
    10021 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
    10081 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
    10141 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
    10201 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
    10261 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
    10321 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
    10381 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
    10441 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
    10501 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
    10561 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
    10621 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
    10681 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
    10741 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
    10801 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
    10861 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
    10921 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
    10981 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
    11041 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
    11101 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
    11161 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
    11221 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
    11281 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
    11341 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
    11401 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
    11461 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
    11521 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
    11581 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
    11641 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg t
//
