LOCUS       sequence-175044-       11707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For cloning and expression of sgRNA together with expression of Cas9
            and TagRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11707)
  AUTHORS   Yumlu S, Bashir S, Stumm J, Kuhn R
  TITLE     Efficient Gene Editing of Human Induced Pluripotent Stem Cells Using
            CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2019;1961:137-151. doi: 
            10.1007/978-1-4939-9170-9_10.
  PUBMED    30912045
REFERENCE   2  (bases 1 to 11707)
  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: "Methods Mol
            Biol."; date: "2019"; volume: "1961:137-151. doi"; pages: " 
            10.1007/978-1-4939-9170-9_10"
COMMENT     Sequence Label: pU6-(BbsI)sgRNA_CAG-Cas9-bpA_EF1-TagRFP
FEATURES             Location/Qualifiers
     source          1..11707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        88..336
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     88..108
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     259..278
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        355..430
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        492..871
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        873..1150
                     /label=chicken beta-actin promoter
     intron          1151..2162
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2086..2108
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2175..2194
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2175..2193
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2198..2207
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2204..6307
                     /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             2204..6307
                     /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             6311..6337
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6317..6343
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             6356..6376
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6383..6403
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6427..6443)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    6463..6687
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        6735..7912
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6965..7903
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7860..7880
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      7913..7922
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7919..8632
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(8658..8674)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    8694..8918
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(8956..8973)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8956..8972)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8965..8987)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(9113..9568)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9250..9271)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     9462..9481
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        9595..9699
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9700..9768,9769..10560)
                     /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(9918..9937)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10731..11319
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11220..11239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11473..11490
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11607..11628
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11643..11648,11649..11666,11667..11673)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11681..11697
                     /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     join(11686..11707,1)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(11705..11707,1..14)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(11705..11707,1..14)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gaaacagcta tgaccatgag gatagggcga attggggccg ctctagaact agtggccccg
       61 cgggccttgg cgcgcccccc cttcaccgag ggcctatttc ccatgattcc ttcatatttg
      121 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
      181 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
      241 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
      301 ttggctttat atatcttgtg gaaaggacga aacaccgggt cttcgagaag acctgtttta
      361 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      421 gagtcggtgc tttttttgat ccaagggtgg gcgcgccaag gcccgcgggg ccactagttc
      481 tagccggatt cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      541 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      601 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      661 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      721 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      781 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      841 catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac
      901 tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt
      961 ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg
     1021 cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct
     1081 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc
     1141 gcggcgggcg ggagtcgctg cgtcgcgcct tcgccccgtg ccccgctccg ccgccgcctc
     1201 gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg
     1261 cccttctcct ccgggctgta attagcgctt ggtttaatga cggctcgttt cttttctgtg
     1321 gctgcgtgaa agccttaaag ggctccggga gggccctttg tgcggggggg agcggctcgg
     1381 ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggcccgcgc tgcccggcgg
     1441 ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc cgcgtgtgcg cgaggggagc
     1501 gcggccgggg gcggtgcccc gcggtgcggg ggggctgcga ggggaacaaa ggctgcgtgc
     1561 ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc ggcggtcggg ctgtaacccc
     1621 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
     1681 gcggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
     1741 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggccccgga
     1801 gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg
     1861 agagggcgca gggacttcct ttgtcccaaa tctggcggag ccgaaatctg ggaggcgccg
     1921 ccgcaccccc tctagcgggc gcgggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     1981 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccatctcca gcctcggggc
     2041 tgccgcaggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     2101 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     2161 agatccttaa ttaataatac gactcactat aggggccgcc accatggaca agaagtacag
     2221 catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa
     2281 ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa
     2341 cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag
     2401 aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt
     2461 cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag agtccttcct
     2521 ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt
     2581 ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac
     2641 cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg
     2701 ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat
     2761 ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt
     2821 ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat
     2881 cgcccagctg cccggcgaga agaagaatgg cctgttcggc aacctgattg ccctgagcct
     2941 gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct
     3001 gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta
     3061 cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct
     3121 gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga
     3181 cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa
     3241 gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg
     3301 agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac
     3361 cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga
     3421 caacggcagc atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca
     3481 ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt
     3541 ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac
     3601 cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc
     3661 ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa
     3721 ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa
     3781 agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa
     3841 ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga
     3901 ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg
     3961 gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt
     4021 cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt
     4081 tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa
     4141 agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct
     4201 gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga
     4261 cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga
     4321 ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa
     4381 tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga
     4441 gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga
     4501 gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga
     4561 gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct
     4621 gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca
     4681 ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt
     4741 tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa
     4801 gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct
     4861 gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg
     4921 cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca
     4981 gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa
     5041 tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt
     5101 ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga
     5161 cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag
     5221 cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga
     5281 gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt
     5341 caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa
     5401 cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt
     5461 gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag
     5521 caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg
     5581 ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt
     5641 ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat
     5701 caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg
     5761 ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct
     5821 ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact
     5881 ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa
     5941 gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct
     6001 ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa
     6061 tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc
     6121 cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta
     6181 ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac
     6241 cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg
     6301 aggcgacgcc tatccctatg acgtgcccga ttatgccagc ctgggcagcg gctcccccaa
     6361 gaaaaaacgc aaggtggaag atcctaagaa aaagcggaaa gtggactgaa cgcgtaaatg
     6421 attgcagatc cactagttct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     6481 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     6541 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     6601 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc
     6661 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tgggctcgag
     6721 gctagagcgg ccgcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     6781 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
     6841 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     6901 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     6961 acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg
     7021 cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt
     7081 tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg
     7141 agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc
     7201 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac
     7261 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc
     7321 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag
     7381 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc
     7441 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc
     7501 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag
     7561 ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa
     7621 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc
     7681 caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg
     7741 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg
     7801 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct
     7861 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gagccaccat
     7921 ggtgtctaag ggcgaagagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     7981 caccgtgaac aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     8041 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     8101 cctggctacc agcttcatgt acggcagcag aaccttcatc aaccacaccc agggcatccc
     8161 cgacttcttt aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     8221 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     8281 caacgtcaag atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac
     8341 actcggctgg gaggccaaca ccgagatgct gtaccccgct gacggcggcc tggaaggcag
     8401 aagcgacatg gccctgaagc tcgtgggcgg gggccacctg atctgcaact tcaagaccac
     8461 atacagatcc aagaaacccg ctaagaacct caagatgccc ggcgtctact atgtggacca
     8521 cagactggaa agaatcaagg aggccgacaa agagacctac gtcgagcagc acgaggtggc
     8581 tgtggccaga tactgcgacc tccctagcaa actggggcac aaacttaatt gagcgatcgc
     8641 acgcgtaaat gattgcagat ccactagttc tagagctcgc tgatcagcct cgactgtgcc
     8701 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     8761 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     8821 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     8881 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     8941 ctgggggcgg ccgccactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     9001 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     9061 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta
     9121 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     9181 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     9241 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     9301 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     9361 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     9421 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     9481 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     9541 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     9601 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     9661 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     9721 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     9781 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     9841 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     9901 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     9961 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
    10021 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
    10081 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
    10141 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
    10201 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
    10261 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
    10321 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
    10381 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
    10441 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
    10501 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
    10561 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
    10621 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
    10681 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
    10741 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
    10801 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
    10861 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
    10921 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
    10981 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
    11041 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
    11101 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
    11161 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
    11221 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
    11281 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
    11341 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
    11401 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
    11461 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
    11521 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
    11581 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
    11641 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
    11701 cacacag
//
