LOCUS       Exported               15470 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  all-in-one-inducible Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    pAAVS1-tet-iCas9-BFP2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15470)
  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 15470)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..15470
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1..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        1944..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             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             8076..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"
     primer_bind     complement(8118..8139)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     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    complement(9074..9107)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     enhancer        9192..9495
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          9742..10758
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     10682..10704
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     10766..10785
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             10824..11573
                     /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    11582..11806
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     12121..12140
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    12139..12194
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     12240..12259
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(13147..13203)
                     /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    13227..13252
                     /label=SA
                     /note="splice acceptor site"
     misc_feature    13266..14069
                     /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   14535..14597
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(14727..14744)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(14727..14743)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14736..14758)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      14885..15340
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(14972..14991)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     15182..15203
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        15366..15470
                     /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 aatactgtcc 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 aacccctgta
     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 gggggggacg gggcagggcg
    10681 gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc
    10741 ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt
    10801 ggcaaagaat tgcggccgcc accatgtcta gactggacaa gagcaaagtc ataaactctg
    10861 ctctggaatt actcaatgga gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa
    10921 agctgggagt tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg
    10981 atgccctgcc aatcgagatg ctggacaggc atcataccca ctcctgcccc ctggaaggcg
    11041 agtcatggca agactttctg cggaacaacg ccaagtcata ccgctgtgct ctcctctcac
    11101 atcgcgacgg ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc
    11161 tggaaaatca gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg
    11221 ctctgtccgc cgtgggccac tttacactgg gctgcgtatt ggaggaacag gagcatcaag
    11281 tagcaaaaga ggaaagagag acacctacca ccgattctat gcccccactt ctgaaacaag
    11341 caattgagct gttcgaccgg cagggagccg aacctgcctt ccttttcggc ctggaactaa
    11401 tcatatgtgg cctggagaaa cagctaaagt gcgaaagcgg cgggccgacc gacgcccttg
    11461 acgattttga cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc
    11521 tgcctgctga cgctcttgac gattttgacc ttgacatgct ccccgggtca taggtttaaa
    11581 cctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
    11641 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
    11701 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
    11761 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctta aggatctcca
    11821 taagagaaga gggacagcta tgactgggag tagtcaggag aggaggaaaa atctggctag
    11881 taaaacaggt aaggaaaatt ttagggatgt taaagaaaaa aataacacaa aacaaaatat
    11941 aaaaaaaatc taacctcaag tcaaggcttt tctatggaat aaggaatgga cagcaggggg
    12001 ctgtttcata tactgatgac ctctttatag ccaacctttg ttcatggcag ccagcatatg
    12061 ggcatatgtt gccaaactct aaaccaaata ctcattctga tgttttaaat gatttgccct
    12121 cccatatgtc cttccgagtg agagacacaa aaaattccaa cacactattg caatgaaaat
    12181 aaatttcctt tattagccag aagtcagatg ctcaaggggc ttcatgatgt ccccataatt
    12241 tttggcagag ggaaaaagat cacagtggta tttgtgagcc agggcattgg ccacaccagc
    12301 caccaccttc tgataggcag cctgcacctg aggagtgtta acctattaaa agaactcatc
    12361 cagcagtctg taaaaagcaa tcctctgtga atcaggtgcg gcaatcccat acagcaccag
    12421 gaacctatcg gcccattccc cgcccagttc ctctgcaatg tctcttgtgg ccagggcgat
    12481 gtcctggtat ctgtcggcga cgcccagtcg tccacaatca ataaacccac tgaatctgcc
    12541 attctccacc atgatgttgg gcaggcaagc gtcgccatgg gtgaccacca gatcttctcc
    12601 gtctggcatt ctggctttca gccgtgcaaa cagctctgca ggagccagcc cctggtgttc
    12661 ctcatccaga tcgtcctggt ccaccagccc ggcctccatt cgggttcgag ccctctcaat
    12721 gcgatgttta gcctggtgat cgaaagggca ggtggcgggg tccagagtat gcagtctccg
    12781 catggcatct gccatgatgg agaccttctc agctggtgcc aggtgggagc tcagcaggtc
    12841 ctgtcctggc acttctccca gcagcagcca atctcgcccg gcctcagtga ccacgtccag
    12901 gactgcagca caaggcactc ctgtggtggc cagccagctc aggcgggctg cttcatcctg
    12961 cagctcgttc agtgctcctg acaggtctgt cttgacaaac agcacggggc gcccctgagc
    13021 actcagccgg aacacggctg cgtcagagca tccaatagtc tgctgtgccc aatcgtatcc
    13081 aaacagtctt tcgacccaag cggctggtga cccggcgtgc agtccgtcct gctcaatcat
    13141 gatatcagga ccggggtttt cttccacgtc tcctgcttgc tttaacagag agaagttcgt
    13201 ggcaccagag ccagatctct cgaggccctg tgggaggaag agaagaggtc agaagcttga
    13261 cgcgtgcccc actgtggggt ggaggggaca gataaaagta cccagaacca gagccacatt
    13321 aaccggccct gggaatataa ggtggtccca gctcggggac acaggatccc tggaggcagc
    13381 aaacatgctg tcctgaagtg gacatagggg cccgggttgg aggaagaaga ctagctgagc
    13441 tctcggaccc ctggaagatg ccatgacagg gggctggaag agctagcaca gactagagag
    13501 gtaagggggg taggggagct gcccaaatga aaggagtgag aggtgacccg aatccacagg
    13561 agaacggggt gtccaggcaa agaaagcaag aggatggaga ggtggctaaa gccagggaga
    13621 cggggtactt tggggttgtc cagaaaaacg gtgatgatgc aggcctacaa gaaggggagg
    13681 cgggacgcaa gggagacatc cgtcggagaa ggccatccta agaaacgaga gatggcacag
    13741 gccccagaag gagaaggaaa agggaaccca gcgagtgaag acggcatggg gttgggtgag
    13801 ggaggagaga tgcccggaga ggacccagac acggggagga tccgctcaga ggacatcacg
    13861 tggtgcagcg ccgagaagga agtgctccgg aaagagcatc cttgggcagc aacacagcag
    13921 agagcaaggg gaagagggag tggaggaaga cggaacctga aggaggcggc agggaaggat
    13981 ctgggccagc cgtagaggtg acccaggcca caagctgcag acagaaagcg gcacaggccc
    14041 aggggagaga atgctggtca gagaaagcag gtaccaaggg cgaattccag cacactggcg
    14101 gccgttacta gtaggccttg 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 ttttttttta ataaataaat aaacataaat aaattgtttg ttgaatttat tattagtatg
    14461 taagtgtaaa tataataaaa cttaatatct attcaaatta ataaataaac ctcgatatac
    14521 agaccgataa aacacatgcg tcaattttac gcatgattat ctttaacgta cgtcacaata
    14581 tgattatctt tctagggtta atctagctgc gtgttctgca gcgtgtcgag catcttcatc
    14641 tgctccatca cgctgtaaaa cacatttgca ccgcgagtct gcccgtcctc cacgggttca
    14701 aaaacgtgaa tgaacgaggc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
    14761 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
    14821 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
    14881 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
    14941 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
    15001 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
    15061 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
    15121 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
    15181 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
    15241 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
    15301 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
    15361 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
    15421 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
//
