LOCUS       sequence-248941-       15469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  all-in-one-inducible Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15469)
  AUTHORS   Mair B, Tomic J, Masud SN, Tonge P, Weiss A, Usaj M, Tong AHY, Kwan 
            JJ, Brown KR, Titus E, Atkins M, Chan KSK, Munsie L, Habsid A, Han 
            H, Kennedy M, Cohen B, Keller G, Moffat J
  TITLE     Essential Gene Profiles for Human Pluripotent Stem Cells Identify 
            Uncharacterized Genes and Substrate Dependencies.
  JOURNAL   Cell Rep. 2019 Apr 9;27(2):599-615.e12. doi: 
            10.1016/j.celrep.2019.02.041.
  PUBMED    30970261
REFERENCE   2  (bases 1 to 15469)
  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"; doi: 
            "10.1016/j.celrep.2019.02"; journalName: "Cell Rep"; date: 
            "2019-04-9- 9"; volume: "27"; issue: "2"; pages: "599-615.e12"
COMMENT     Sequence Label: pAAVS1-tet-iCas9-BFP2
FEATURES             Location/Qualifiers
     source          1..15469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..69,70..861)
                     /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(219..238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1032..1620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1521..1540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1774..1791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1908..1929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1944..1949,1950..1967,1968..1974)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1982..1998
                     /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     1987..2009
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2006..2022
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2006..2022
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2513..2547
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     misc_feature    2557..3393
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        3422..3789
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    3430..3448
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3466..3484
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3502..3520
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3538..3556
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3574..3592
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3610..3628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3646..3664
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3807..7910
                     /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             3807..7910
                     /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             7911..7958
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7959..7982
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7992..8048
                     /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             8055..8075
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(8076..8078,8079..8789)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIKV
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLLE
                     FRTAAGITLGMDELYK"
     CDS             8790..8837
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    8849..9073
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    9074..9107
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        9192..9495
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          9742..10759
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     10683..10705
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     10767..10786
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             10825..11574
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPGS"
     polyA_signal    11583..11807
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     12122..12141
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    12140..12195
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     12241..12260
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(13148..13204)
                     /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"
     misc_feature    13228..13253
                     /label=SA
                     /note="splice acceptor site"
     misc_feature    13267..14070
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     repeat_region   14534..14596
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(14726..14742)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14735..14757)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      14884..15339
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(14971..14990)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     15181..15202
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        15365..15469
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
       61 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
      121 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
      181 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
      241 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
      301 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
      361 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
      421 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
      481 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
      541 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
      601 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
      661 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
      721 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
      781 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
      841 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
      901 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
      961 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1021 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1081 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1141 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     1201 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1261 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1321 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1381 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1441 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1501 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1561 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1621 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     1681 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     1741 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     1801 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     1861 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     1921 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     1981 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
     2041 gcccgccggg taactcacgg ggtatccatg tccatttctg cggcatccag ccaggatacc
     2101 cgtcctcgct gacgtaatat cccagcgccg caccgctgtc attaatctgc acaccggcac
     2161 ggcagttccg gctgtcgccg gtattgttcg ggttgctgat gcgcttcggg ctgaccatcc
     2221 ggaactgtgt ccggaaaagc cgcgacgaac tggtatccca ggtggcctga acgaacagtt
     2281 caccgttaaa ggcgtgcatg gccacacctt cccgaatcat catggtaaac gtgcgttttc
     2341 gctcaacgtc aatgcagcag cagtcatcct cggcaaactc tttccatgcc gcttcaacct
     2401 cgcgggaaaa ggcacgggct tcttcctccc cgatgcccag atagcgccag cttgggcgat
     2461 gactgagccg gaaaaaagac ccgacgatat gatcctgatg cagctagatt aaccctagaa
     2521 agatagtctg cgtaaaattg acgcatgcat ctcgagagag cagagccagg aacacctgta
     2581 gggaaggggc aggagagcca ggggcatgag atggtggacg aggaaggggg acagggaagc
     2641 ctgagcgcct ctcctgggct tgccaaggac tcaaacccag aagcccagag cagggcctta
     2701 gggaagcggg accctgctct gggcggagga atatgtccca gatagcactg gggactcttt
     2761 aaggaaagaa ggatggagaa agagaaaggg agtagaggcg gccacgacct ggtgaacacc
     2821 taggacgcac cattctcaca aagggagttt tccacacgga cacccccctc ctcaccacag
     2881 ccctgccagg acggggctgg ctactggcct tatctcacag gtaaaactga cgcacggagg
     2941 aacaatataa attggggact agaaaggtga agagccaaag ttagaactca ggaccaactt
     3001 attctgattt tgtttttcca aactgcttct cctcttggga agtgtaagga agctgcagca
     3061 ccaggatcag tgaaacgcac cagacagccg cgtcagagca gctcaggttc tgggagaggg
     3121 tagcgcaggg tggccactga gaaccgggca ggtcacgcat cccccccttc cctcccaccc
     3181 cctgccaagc tctccctccc aggatcctct ctggctccat cgtaagcaaa ccttagaggt
     3241 tctggcaagg agagagatgg ctccaggaaa tgggggtgtg tcaccagata aggaatctgc
     3301 ctaacaggag gtgggggtta gacccaatat caggagacta ggaaggagga ggcctaagga
     3361 tggggctttt ctgtcaccaa tcctgtccct agtagtgtac atgccagaac atttctccct
     3421 cgagtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
     3481 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
     3541 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
     3601 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
     3661 gagaacgtat aagctttgct tatgtaaacc agggcgccta taaaagagtg ctgatttttt
     3721 gagtaaactt caattccaca acacttttgt cttataccaa ctttccgtac cacttcctac
     3781 cctcgtaaag ccgggcgcgc cccaccatgg acaagaagta cagcatcggc ctggacatcg
     3841 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     3901 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     3961 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     4021 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     4081 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     4141 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     4201 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     4261 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     4321 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     4381 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     4441 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     4501 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     4561 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     4621 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     4681 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     4741 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     4801 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     4861 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     4921 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     4981 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     5041 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     5101 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     5161 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     5221 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     5281 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     5341 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     5401 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     5461 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     5521 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     5581 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     5641 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     5701 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     5761 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     5821 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     5881 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     5941 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     6001 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     6061 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     6121 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     6181 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     6241 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     6301 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     6361 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     6421 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     6481 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     6541 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     6601 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     6661 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     6721 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     6781 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     6841 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     6901 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     6961 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     7021 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     7081 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     7141 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     7201 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     7261 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     7321 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     7381 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     7441 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     7501 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     7561 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     7621 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     7681 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     7741 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     7801 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     7861 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aagcgacctg
     7921 ccgccacaaa gaaggctgga caggctaaga agaagaaaga ttacaaagac gatgacgata
     7981 agggatccgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc
     8041 ctggaccggt cgaccctaag aaaaagcgga aggtggtgag caagggcgag gagctgttca
     8101 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     8161 tgaggggcga gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca
     8221 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgagc cacggcgtgc
     8281 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     8341 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcacc tacaagaccc
     8401 gcgccgaggt gaagttcgag ggcgacaccc tagtgaaccg catcgagctg aagggcgtcg
     8461 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccaca
     8521 acatctatat catggccgtc aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     8581 acaacgtgga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     8641 gcgacggccc cgtgctgctg cccgacagcc actacctgag cacccagtcc gtgctgagca
     8701 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttccgcacc gccgccggga
     8761 tcactctcgg catggacgag ctgtacaaga agcgtcctgc tgctactaag aaagctggtc
     8821 aagctaagaa aaagaaataa ttaattaact gtgccttcta gttgccagcc atctgttgtt
     8881 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     8941 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     9001 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     9061 gtgggctcta tggataactt cgtataatgt atgctatacg aagttatgcc cgggcaagga
     9121 tctgcgatcg ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     9181 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     9241 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     9301 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     9361 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     9421 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     9481 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     9541 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     9601 gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag
     9661 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
     9721 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
     9781 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
     9841 gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt
     9901 tcttttctgt ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg
     9961 agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg
    10021 ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg
    10081 cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac
    10141 aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc
    10201 gggctgcaac cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt
    10261 gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag
    10321 gtgggggtgc cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc
    10381 ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta
    10441 tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa
    10501 tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc
    10561 aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct
    10621 ctccagcctc ggggctgtcc gcggggggac ggctgccttc ggggggggac ggggcagggc
    10681 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc
    10741 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt
    10801 tggcaaagaa ttgcggccgc caccatgtct agactggaca agagcaaagt cataaactct
    10861 gctctggaat tactcaatgg agtcggtatc gaaggcctga cgacaaggaa actcgctcaa
    10921 aagctgggag ttgagcagcc taccctgtac tggcacgtga agaacaagcg ggccctgctc
    10981 gatgccctgc caatcgagat gctggacagg catcataccc actcctgccc cctggaaggc
    11041 gagtcatggc aagactttct gcggaacaac gccaagtcat accgctgtgc tctcctctca
    11101 catcgcgacg gggctaaagt gcatctcggc acccgcccaa cagagaaaca gtacgaaacc
    11161 ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct ccctggagaa cgcactgtac
    11221 gctctgtccg ccgtgggcca ctttacactg ggctgcgtat tggaggaaca ggagcatcaa
    11281 gtagcaaaag aggaaagaga gacacctacc accgattcta tgcccccact tctgaaacaa
    11341 gcaattgagc tgttcgaccg gcagggagcc gaacctgcct tccttttcgg cctggaacta
    11401 atcatatgtg gcctggagaa acagctaaag tgcgaaagcg gcgggccgac cgacgccctt
    11461 gacgattttg acttagacat gctcccagcc gatgcccttg acgactttga ccttgatatg
    11521 ctgcctgctg acgctcttga cgattttgac cttgacatgc tccccgggtc ataggtttaa
    11581 acctgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
    11641 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
    11701 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
    11761 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt aaggatctcc
    11821 ataagagaag agggacagct atgactggga gtagtcagga gaggaggaaa aatctggcta
    11881 gtaaaacagg taaggaaaat tttagggatg ttaaagaaaa aaataacaca aaacaaaata
    11941 taaaaaaaat ctaacctcaa gtcaaggctt ttctatggaa taaggaatgg acagcagggg
    12001 gctgtttcat atactgatga cctctttata gccaaccttt gttcatggca gccagcatat
    12061 gggcatatgt tgccaaactc taaaccaaat actcattctg atgttttaaa tgatttgccc
    12121 tcccatatgt ccttccgagt gagagacaca aaaaattcca acacactatt gcaatgaaaa
    12181 taaatttcct ttattagcca gaagtcagat gctcaagggg cttcatgatg tccccataat
    12241 ttttggcaga gggaaaaaga tcacagtggt atttgtgagc cagggcattg gccacaccag
    12301 ccaccacctt ctgataggca gcctgcacct gaggagtgtt aacctattaa aagaactcat
    12361 ccagcagtct gtaaaaagca atcctctgtg aatcaggtgc ggcaatccca tacagcacca
    12421 ggaacctatc ggcccattcc ccgcccagtt cctctgcaat gtctcttgtg gccagggcga
    12481 tgtcctggta tctgtcggcg acgcccagtc gtccacaatc aataaaccca ctgaatctgc
    12541 cattctccac catgatgttg ggcaggcaag cgtcgccatg ggtgaccacc agatcttctc
    12601 cgtctggcat tctggctttc agccgtgcaa acagctctgc aggagccagc ccctggtgtt
    12661 cctcatccag atcgtcctgg tccaccagcc cggcctccat tcgggttcga gccctctcaa
    12721 tgcgatgttt agcctggtga tcgaaagggc aggtggcggg gtccagagta tgcagtctcc
    12781 gcatggcatc tgccatgatg gagaccttct cagctggtgc caggtgggag ctcagcaggt
    12841 cctgtcctgg cacttctccc agcagcagcc aatctcgccc ggcctcagtg accacgtcca
    12901 ggactgcagc acaaggcact cctgtggtgg ccagccagct caggcgggct gcttcatcct
    12961 gcagctcgtt cagtgctcct gacaggtctg tcttgacaaa cagcacgggg cgcccctgag
    13021 cactcagccg gaacacggct gcgtcagagc atccaatagt ctgctgtgcc caatcgtatc
    13081 caaacagtct ttcgacccaa gcggctggtg acccggcgtg cagtccgtcc tgctcaatca
    13141 tgatatcagg accggggttt tcttccacgt ctcctgcttg ctttaacaga gagaagttcg
    13201 tggcaccaga gccagatctc tcgaggccct gtgggaggaa gagaagaggt cagaagcttg
    13261 acgcgtgccc cactgtgggg tggaggggac agataaaagt acccagaacc agagccacat
    13321 taaccggccc tgggaatata aggtggtccc agctcgggga cacaggatcc ctggaggcag
    13381 caaacatgct gtcctgaagt ggacataggg gcccgggttg gaggaagaag actagctgag
    13441 ctctcggacc cctggaagat gccatgacag ggggctggaa gagctagcac agactagaga
    13501 ggtaaggggg gtaggggagc tgcccaaatg aaaggagtga gaggtgaccc gaatccacag
    13561 gagaacgggg tgtccaggca aagaaagcaa gaggatggag aggtggctaa agccagggag
    13621 acggggtact ttggggttgt ccagaaaaac ggtgatgatg caggcctaca agaaggggag
    13681 gcgggacgca agggagacat ccgtcggaga aggccatcct aagaaacgag agatggcaca
    13741 ggccccagaa ggagaaggaa aagggaaccc agcgagtgaa gacggcatgg ggttgggtga
    13801 gggaggagag atgcccggag aggacccaga cacggggagg atccgctcag aggacatcac
    13861 gtggtgcagc gccgagaagg aagtgctccg gaaagagcat ccttgggcag caacacagca
    13921 gagagcaagg ggaagaggga gtggaggaag acggaacctg aaggaggcgg cagggaagga
    13981 tctgggccag ccgtagaggt gacccaggcc acaagctgca gacagaaagc ggcacaggcc
    14041 caggggagag aatgctggtc agagaaagca ggtaccaagg gcgaattcca gcacactggc
    14101 ggccgttact agtggccgtg gaggcctttt ccccgtatcc ccccaggtgt ctgcaggctc
    14161 aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg
    14221 ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc
    14281 cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt
    14341 gggggggggc tgtccctcat aaaagttttg ttactttata gaagaaattt tgagtttttg
    14401 ttttttttaa taaataaata aacataaata aattgtttgt tgaatttatt attagtatgt
    14461 aagtgtaaat ataataaaac ttaatatcta ttcaaattaa taaataaacc tcgatataca
    14521 gaccgataaa acacatgcgt caattttaca catgattatc tttaacgtac gtcacaatat
    14581 gattatcttt ctagggttaa tctagctgcg tgttctgcag cgtgtcgagc atcttcatct
    14641 gctccatcac gctgtaaaac acatttgcac cgcgagtctg cccgtcctcc acgggttcaa
    14701 aaacgtgaat gaacgaggcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
    14761 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
    14821 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
    14881 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
    14941 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
    15001 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
    15061 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
    15121 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
    15181 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
    15241 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
    15301 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
    15361 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
    15421 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagt
//
