LOCUS       sequence-362612-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets CSF1R 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_CSF1R
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    15..31
                     /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     20..42
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     39..55
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     39..55
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     74..94
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        76..94
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        122..348
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             349..529
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    576..701
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1194..1427
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1612..1656
                     /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    1837..1954
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2143..2354
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2373..2382
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2379..6482
                     /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             2379..6482
                     /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             6483..6530
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6531..6554
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6564..6620
                     /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             6621..7220
                     /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(6621..6640)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7117..7137
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7236..7824
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7289..7309)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7707..7718)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7980..8228
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7980..8000
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8151..8170
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8250..8325
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8378..8558
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8636..8757
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8673..8692)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8727..8746
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8797..8932
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8952..8971)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8953..8971)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8981..8998)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8981..8997)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8990..9012)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9139..9594
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9226..9245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9436..9457
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9620..9724
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9725..9793,9794..10585)
                     /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(9943..9962)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10756..11344
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11245..11264
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11498..11515
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11632..11653
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11668..11673,11674..11691,1..7)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
       61 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
      121 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      181 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      241 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      301 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      361 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      421 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      481 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      541 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      601 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      661 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      721 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      781 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
      841 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
      901 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
      961 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1021 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1081 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1141 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1201 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1261 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1321 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1381 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1441 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1501 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1561 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1621 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1681 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1741 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1801 cagaatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg
     1861 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga
     1921 attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga
     1981 tccactttgg cgccggctcg agggggcccg ggtgcaaaga tggataaagt tttaaacaga
     2041 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgt
     2101 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc
     2161 ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt
     2221 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     2281 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     2341 ccgccagaac acaggaccgg ttctagagcg ctgccaccat ggacaagaag tacagcatcg
     2401 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     2461 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     2521 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     2581 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     2641 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     2701 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     2761 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     2821 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     2881 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     2941 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     3001 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     3061 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     3121 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     3181 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     3241 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     3301 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     3361 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     3421 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     3481 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     3541 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     3601 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     3661 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     3721 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     3781 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     3841 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     3901 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     3961 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     4021 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     4081 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     4141 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     4201 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     4261 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     4321 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     4381 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     4441 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     4501 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     4561 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     4621 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     4681 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     4741 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     4801 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     4861 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     4921 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     4981 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     5041 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     5101 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     5161 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     5221 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     5281 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     5341 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     5401 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     5461 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     5521 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     5581 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     5641 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     5701 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     5761 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     5821 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     5881 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     5941 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     6001 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     6061 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     6121 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     6181 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     6241 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     6301 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     6361 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     6421 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     6481 acaagcgacc tgccgccaca aagaaggctg gacaggctaa gaagaagaaa gattacaaag
     6541 acgatgacga taagggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg
     6601 tcgaagagaa tcctggaccg atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     6661 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     6721 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     6781 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgccgtgg
     6841 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     6901 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     6961 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc
     7021 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     7081 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     7141 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     7201 cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat tacaaaattt
     7261 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     7321 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     7381 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     7441 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     7501 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     7561 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     7621 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     7681 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     7741 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     7801 tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt
     7861 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg
     7921 aagacaagat ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag
     7981 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     8041 taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     8101 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     8161 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     8221 gaaacaccgg catggcttca ccatccacag ttttagagct agaaatagca agttaaaata
     8281 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt taagcttggc
     8341 gtaactagat cttgagacac tgctttttgc ttgtactggg tctctctggt tagaccagat
     8401 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     8461 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     8521 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca
     8581 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     8641 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     8701 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     8761 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     8821 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     8881 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg
     8941 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     9001 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     9061 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     9121 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     9181 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     9241 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     9301 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     9361 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     9421 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     9481 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     9541 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     9601 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     9661 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     9721 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     9781 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     9841 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     9901 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     9961 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
    10021 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
    10081 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
    10141 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
    10201 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
    10261 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
    10321 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
    10381 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
    10441 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
    10501 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
    10561 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
    10621 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
    10681 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
    10741 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
    10801 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
    10861 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
    10921 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
    10981 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
    11041 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
    11101 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
    11161 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
    11221 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
    11281 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
    11341 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
    11401 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
    11461 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
    11521 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
    11581 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
    11641 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc g
//
