LOCUS       sequence-362774-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system containing a non-targeting control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  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 13013)
  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_NTC3
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(628..1419,1420..1488))
                     /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     1251..1270
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1489..1593)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1668..1687)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1859..2238
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2240..2438
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2393..2413
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2456..2636
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2683..2808
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3301..3534
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3719..3763
                     /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    4061..4178
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4229..4477
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4229..4249
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4400..4419
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4499..4574
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4636..4847
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4866..4875
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4872..8975
                     /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             4872..8975
                     /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             8976..9023
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9024..9047
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             9057..9113
                     /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             9114..9710
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     9607..9627
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9726..10314
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9779..9799)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10197..10208)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10386..10619
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10645..10662)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10651..10875
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10921..11349
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11008..11027)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11218..11239
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11358..11378)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11363..11692
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11543..11678
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11605..11624
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11740..11787
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11806..12180
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    12310..12431
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12347..12366)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12401..12420
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12480..12496)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12480..12496)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12493..12515)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12504..12520
                     /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)."
     promoter        complement(join(12528..12534,12535..12552,12553..12558))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12573..12594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12711..12728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(12882..13013,1..457))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12962..12981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
       61 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
      121 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
      181 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
      241 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
      301 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
      361 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
      421 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
      481 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
      541 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
      601 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
      661 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
      721 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
      781 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
      841 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
      901 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
      961 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     1021 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     1081 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     1141 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     1201 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     1261 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     1321 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     1381 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     1441 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     1501 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     1561 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     1621 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     1681 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     1741 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     1801 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
     1861 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     1921 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     1981 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     2041 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     2101 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     2161 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     2221 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     2281 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     2341 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     2401 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
     2461 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     2521 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     2581 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     2641 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     2701 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     2761 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     2821 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     2881 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     2941 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     3001 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     3061 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     3121 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     3181 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     3241 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     3301 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     3361 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     3421 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     3481 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     3541 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     3601 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     3661 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     3721 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     3781 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     3841 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     3901 atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga
     3961 agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg
     4021 tgcgccaatt ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     4081 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     4141 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     4201 gagatccagt ttggttaatt aaggtaccga gggcctattt cccatgattc cttcatattt
     4261 gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa
     4321 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     4381 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     4441 cttggcttta tatatcttgt ggaaaggacg aaacaccgaa gtgacggtgt catgcggggt
     4501 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     4561 caccgagtcg gtgctttttt gaattcgcta gctaggtctt gaaaggagtg ggaattggct
     4621 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     4681 gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     4741 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     4801 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggac cggttctaga
     4861 gcgctgccac catggacaag aagtacagca tcggcctgga catcggcacc aactctgtgg
     4921 gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca
     4981 acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg
     5041 aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga
     5101 accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct
     5161 tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc
     5221 ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc
     5281 acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg
     5341 ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg
     5401 acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg
     5461 aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga
     5521 gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc
     5581 tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg
     5641 acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca
     5701 acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt
     5761 ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc
     5821 tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag
     5881 ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga
     5941 acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca
     6001 agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg
     6061 acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg
     6121 gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc
     6181 gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca
     6241 ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga
     6301 acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca
     6361 acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt
     6421 acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc
     6481 ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc
     6541 ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact
     6601 ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc
     6661 tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg
     6721 aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga
     6781 aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca
     6841 ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca
     6901 agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga
     6961 tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg
     7021 gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca
     7081 tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg
     7141 agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca
     7201 gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga
     7261 aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc
     7321 agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg
     7381 atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc
     7441 tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga
     7501 agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt
     7561 tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca
     7621 tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact
     7681 cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca
     7741 ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg
     7801 agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc
     7861 tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg
     7921 acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact
     7981 tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga
     8041 tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg
     8101 gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa
     8161 agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg
     8221 ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc
     8281 ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac
     8341 tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga
     8401 atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca
     8461 agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg
     8521 ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt
     8581 acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc
     8641 tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct
     8701 ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc
     8761 accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca
     8821 atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca
     8881 ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg
     8941 agacacggat cgacctgtct cagctgggag gcgacaagcg acctgccgcc acaaagaagg
     9001 ctggacaggc taagaagaag aaagattaca aagacgatga cgataaggga tccggcgcaa
     9061 caaacttctc tctgctgaaa caagccggag atgtcgaaga gaatcctgga ccgaccgagt
     9121 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     9181 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     9241 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     9301 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     9361 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     9421 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     9481 tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     9541 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     9601 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     9661 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag
     9721 tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     9781 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     9841 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     9901 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     9961 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
    10021 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
    10081 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
    10141 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
    10201 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
    10261 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac
    10321 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
    10381 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
    10441 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
    10501 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
    10561 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
    10621 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
    10681 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
    10741 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
    10801 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
    10861 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
    10921 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
    10981 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
    11041 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
    11101 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
    11161 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
    11221 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
    11281 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
    11341 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
    11401 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
    11461 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
    11521 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
    11581 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
    11641 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
    11701 gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca
    11761 tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc
    11821 agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac
    11881 cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac
    11941 gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg
    12001 gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac
    12061 ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag
    12121 ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga
    12181 cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
    12241 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
    12301 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
    12361 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
    12421 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
    12481 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
    12541 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
    12601 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
    12661 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
    12721 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
    12781 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
    12841 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
    12901 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
    12961 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tcc
//
