LOCUS       sequence-362643-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets KIT 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_KIT
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        8..234
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             235..415
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    462..587
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1080..1313
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1498..1542
                     /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    1723..1840
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2029..2240
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2259..2268
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2265..6368
                     /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             2265..6368
                     /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             6369..6416
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6417..6440
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6450..6506
                     /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             6507..7106
                     /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(6507..6526)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7003..7023
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7122..7710
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7175..7195)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7593..7604)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7866..8114
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7866..7886
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8037..8056
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8136..8211
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8264..8444
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8522..8643
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8559..8578)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8613..8632
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8683..8818
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8838..8857)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8839..8857)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8867..8884)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8867..8883)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8876..8898)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9025..9480
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9112..9131)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9322..9343
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9506..9610
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9611..9679,9680..10471)
                     /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(9829..9848)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10642..11230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11131..11150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11384..11401
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11518..11539
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11554..11559,11560..11577,11578..11584)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11592..11608
                     /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     11597..11619
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11616..11632
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11616..11632
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11651..11671
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11653..11671
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
       61 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
      121 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
      181 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
      241 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      301 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      361 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
      421 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
      481 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
      541 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
      601 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
      661 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
      721 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
      781 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
      841 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
      901 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
      961 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1021 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1081 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1141 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1201 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1261 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1321 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1381 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1441 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1501 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1561 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1621 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     1681 tcgtttcaga atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     1741 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     1801 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     1861 cagagatcca ctttggcgcc ggctcgaggg ggcccgggtg caaagatgga taaagtttta
     1921 aacagagagg aatctttgca gctaatggac cttctaggtc ttgaaaggag tgggaattgg
     1981 ctccgtaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca
     2041 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgatcc ggtgcctaga
     2101 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     2161 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     2221 ggtttgccgc cagaacacag gaccggttct agagcgctgc caccatggac aagaagtaca
     2281 gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
     2341 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
     2401 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
     2461 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     2521 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     2581 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     2641 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     2701 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     2761 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     2821 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     2881 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     2941 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc
     3001 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     3061 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     3121 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     3181 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     3241 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     3301 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg
     3361 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     3421 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     3481 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     3541 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     3601 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     3661 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     3721 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     3781 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca
     3841 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     3901 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     3961 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     4021 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     4081 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     4141 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     4201 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     4261 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     4321 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     4381 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
     4441 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
     4501 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
     4561 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
     4621 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
     4681 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
     4741 aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct
     4801 ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca
     4861 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc
     4921 tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag
     4981 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
     5041 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
     5101 atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
     5161 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
     5221 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
     5281 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
     5341 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
     5401 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
     5461 acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag
     5521 tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
     5581 gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact
     5641 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
     5701 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
     5761 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
     5821 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
     5881 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
     5941 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
     6001 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc
     6061 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
     6121 atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg
     6181 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
     6241 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
     6301 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
     6361 gaggcgacaa gcgacctgcc gccacaaaga aggctggaca ggctaagaag aagaaagatt
     6421 acaaagacga tgacgataag ggatccggcg caacaaactt ctctctgctg aaacaagccg
     6481 gagatgtcga agagaatcct ggaccgatga ccgagtacaa gcccacggtg cgcctcgcca
     6541 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     6601 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     6661 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     6721 ccgtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     6781 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     6841 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc
     6901 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     6961 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     7021 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     7081 gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca
     7141 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     7201 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     7261 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     7321 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     7381 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     7441 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     7501 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     7561 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     7621 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     7681 gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc
     7741 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     7801 ccaacgaaga caagatttca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
     7861 ccagagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     7921 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     7981 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     8041 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     8101 aaggacgaaa caccgtttgt ccaggaactg agcaggtttt agagctagaa atagcaagtt
     8161 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttaag
     8221 cttggcgtaa ctagatcttg agacactgct ttttgcttgt actgggtctc tctggttaga
     8281 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     8341 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     8401 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc
     8461 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     8521 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     8581 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     8641 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg
     8701 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     8761 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     8821 gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     8881 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     8941 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     9001 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     9061 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     9121 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     9181 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     9241 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     9301 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     9361 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     9421 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     9481 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     9541 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     9601 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     9661 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     9721 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     9781 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     9841 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     9901 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     9961 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
    10021 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
    10081 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
    10141 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
    10201 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
    10261 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
    10321 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
    10381 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
    10441 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
    10501 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
    10561 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
    10621 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
    10681 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
    10741 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
    10801 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
    10861 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
    10921 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
    10981 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
    11041 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
    11101 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
    11161 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
    11221 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
    11281 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
    11341 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
    11401 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
    11461 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
    11521 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
    11581 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
    11641 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc a
//
