LOCUS       sequence-490176-       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_NTC1
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(760..1551,1552..1620))
                     /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     1383..1402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1621..1725)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1800..1819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1991..2370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2372..2570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2525..2545
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2588..2768
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2815..2940
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3433..3666
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3851..3895
                     /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    4193..4310
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4361..4609
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4361..4381
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4532..4551
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4631..4706
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4768..4979
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4998..5007
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5004..9107
                     /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             5004..9107
                     /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             9108..9155
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9156..9179
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             9189..9245
                     /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             9246..9842
                     /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     9739..9759
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9858..10446
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9911..9931)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10329..10340)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10518..10751
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10777..10794)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10783..11007
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11053..11481
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11140..11159)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11350..11371
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11490..11510)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11495..11824
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11675..11810
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11737..11756
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11872..11919
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11938..12312
                     /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    12442..12563
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12479..12498)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12533..12552
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12612..12628)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12612..12628)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12625..12647)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12636..12652
                     /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(12660..12666,12667..12684,12685..12690))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12705..12726
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12843..12860)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      841 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      901 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      961 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1021 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1081 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1141 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1201 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1261 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1321 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1381 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1441 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1501 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1561 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1621 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1681 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1741 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     1801 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     1861 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     1921 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     1981 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     2041 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     2101 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     2161 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     2221 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     2281 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     2341 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     2401 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     2461 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     2521 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc
     2581 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     2641 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     2701 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     2761 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
     2821 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
     2881 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
     2941 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
     3001 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
     3061 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     3121 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     3181 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     3241 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg
     3301 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
     3361 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
     3421 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
     3481 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
     3541 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
     3601 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
     3661 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
     3721 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
     3781 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
     3841 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
     3901 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
     3961 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg
     4021 atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga
     4081 aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg
     4141 atcggcactg cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa
     4201 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     4261 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     4321 acagggacag cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat
     4381 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
     4441 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     4501 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     4561 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg aaaaagcttc
     4621 cgcctgatgg gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     4681 ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag
     4741 tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
     4801 agttgggggg aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact
     4861 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
     4921 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
     4981 accggttcta gagcgctgcc accatggaca agaagtacag catcggcctg gacatcggca
     5041 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
     5101 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
     5161 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
     5221 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
     5281 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
     5341 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
     5401 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
     5461 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
     5521 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
     5581 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
     5641 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
     5701 agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca
     5761 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
     5821 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
     5881 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
     5941 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
     6001 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
     6061 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
     6121 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
     6181 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
     6241 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
     6301 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
     6361 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
     6421 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
     6481 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
     6541 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
     6601 gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt
     6661 tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg
     6721 agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca
     6781 cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg
     6841 aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
     6901 aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc
     6961 ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca
     7021 agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact
     7081 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
     7141 tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
     7201 ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc
     7261 ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg
     7321 gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
     7381 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
     7441 tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
     7501 tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg
     7561 acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg
     7621 aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta
     7681 cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata
     7741 aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
     7801 agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag
     7861 tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt
     7921 acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg
     7981 tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact
     8041 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
     8101 ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg
     8161 ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg
     8221 tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga
     8281 atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca
     8341 agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
     8401 gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
     8461 agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca
     8521 gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg
     8581 acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa
     8641 tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
     8701 tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg
     8761 agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga
     8821 tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg
     8881 cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt
     8941 ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc
     9001 ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca
     9061 ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaag cgacctgccg
     9121 ccacaaagaa ggctggacag gctaagaaga agaaagatta caaagacgat gacgataagg
     9181 gatccggcgc aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg
     9241 gaccgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     9301 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     9361 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     9421 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     9481 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     9541 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     9601 agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg
     9661 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     9721 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     9781 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     9841 gaacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     9901 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     9961 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
    10021 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
    10081 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
    10141 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
    10201 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
    10261 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
    10321 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
    10381 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
    10441 ccccgcgtcg actttaagac caatgactta caaggcagct gtagatctta gccacttttt
    10501 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt
    10561 tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
    10621 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
    10681 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
    10741 aatctctagc agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
    10801 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
    10861 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
    10921 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
    10981 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
    11041 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
    11101 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
    11161 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
    11221 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
    11281 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
    11341 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
    11401 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
    11461 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
    11521 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
    11581 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
    11641 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
    11701 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
    11761 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
    11821 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat
    11881 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg
    11941 gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag
    12001 ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg
    12061 gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac
    12121 accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc
    12181 gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg
    12241 tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag
    12301 gagcaggact gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg
    12361 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
    12421 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
    12481 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
    12541 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
    12601 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
    12661 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
    12721 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
    12781 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
    12841 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
    12901 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
    12961 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtt
//
