LOCUS       sequence-261205-        9991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty concatemer vector in which 3 sgRNAs can be inserted; with Cas9
            + Puro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9991)
  AUTHORS   Gaebler AM, Hennig A, Buczolich K, Weitz J, Welsch T, Stange DE, 
            Pape K
  TITLE     Universal and Efficient Electroporation Protocol for Genetic 
            Engineering of Gastrointestinal Organoids.
  JOURNAL   J Vis Exp. 2020 Feb 18;(156). doi: 10.3791/60704.
  PUBMED    32150173
REFERENCE   2  (bases 1 to 9991)
  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: "J Vis 
            Exp."; date: "2020-02-18"; volume: "(156). doi"; pages: " 
            10.3791/60704"
COMMENT     Sequence Label: px459V2.0_Conc3
FEATURES             Location/Qualifiers
     source          1..9991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        783..1031
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1048..1123
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1130..1153
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        1176..1424
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1443..1518
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1524..1550
                     /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="YPYDVPDYA"
     CDS             1524..1550
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        1574..1822
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1843..1918
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1982..2267
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2269..2546
                     /label=chicken beta-actin promoter
     intron          2547..2775
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2787..2796
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2796..2861
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2868..2888
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2913..7013
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2913..7013
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7014..7061
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7077..7130
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             7131..7730
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7131..7150)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(7758..7775)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7764..7971
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7980..8120
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7980..8109
                     /label=AAV2 ITR
     rep_origin      8195..8650
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8282..8301)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8492..8513
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8667..8686)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8786..8808
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8846..8864)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8932..9036
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9037..9105,9106..9897)
                     /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(9255..9274)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgtccag ccctcactcc ttctctaggc gccggaatta gatctccaag gtcgggcagg
      781 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
      841 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
      901 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
      961 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     1021 gacgaaacac cgggtcttcg agaagacgtt ttagagctag aaatagcaag ttaaaataag
     1081 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg actacaagga
     1141 cgacgatgac aaggatctcc aaggtcgggc aggaagaggg cctatttccc atgattcctt
     1201 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     1261 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     1321 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     1381 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagtc ttcgagaaga
     1441 cagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     1501 gtggcaccga gtcggtgctt ttttacccct acgacgtgcc cgactacgcc ggatctccaa
     1561 ggtcgggcag gaagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     1621 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     1681 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     1741 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     1801 cttgtggaaa ggacgaaaca ccggaagtct tcgagaagac aagttttaga gctagaaata
     1861 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     1921 ttttatggca tcaatgcaga agctgatctc agaggaggac ctgggatcct ctagaggtac
     1981 ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     2041 tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     2101 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     2161 acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc ttatgggact
     2221 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
     2281 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
     2341 ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag
     2401 gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
     2461 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
     2521 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc
     2581 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
     2641 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg
     2701 tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa
     2761 atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact
     2821 acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc
     2881 ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca
     2941 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     3001 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     3061 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     3121 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     3181 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     3241 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     3301 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     3361 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     3421 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     3481 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     3541 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     3601 gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca
     3661 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     3721 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     3781 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     3841 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     3901 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     3961 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     4021 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     4081 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     4141 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     4201 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     4261 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     4321 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     4381 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     4441 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     4501 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     4561 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     4621 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     4681 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     4741 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     4801 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     4861 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     4921 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     4981 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     5041 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     5101 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     5161 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     5221 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     5281 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     5341 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     5401 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact
     5461 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     5521 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     5581 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     5641 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     5701 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     5761 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     5821 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     5881 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     5941 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     6001 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     6061 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     6121 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     6181 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     6241 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     6301 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     6361 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     6421 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     6481 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     6541 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     6601 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     6661 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     6721 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     6781 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     6841 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     6901 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     6961 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacaaaaggc
     7021 cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa ggaattcggc agtggagagg
     7081 gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca atgaccgagt
     7141 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     7201 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     7261 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     7321 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     7381 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     7441 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     7501 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     7561 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     7621 cgccccacaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     7681 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga gaattctaac
     7741 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     7801 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     7861 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     7921 gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca
     7981 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     8041 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     8101 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     8161 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     8221 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     8281 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     8341 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     8401 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     8461 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     8521 ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     8581 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     8641 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     8701 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     8761 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     8821 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     8881 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     8941 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     9001 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     9061 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     9121 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     9181 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     9241 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     9301 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     9361 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     9421 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     9481 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     9541 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     9601 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     9661 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     9721 attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg
     9781 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     9841 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     9901 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     9961 aggatctagg tgaagatcct ttttgataat c
//
